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Volume 2, Issue 22012

ISSN: 2159-8967www.AFABjournal.com

78 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 79

Sooyoun Ahn University of Florida, USA

Walid Q. AlaliUniversity of Georgia, USA

Kenneth M. Bischoff NCAUR, USDA-ARS, USA

Claudia S. Dunkley University of Georgia, USA

Lawrence GoodridgeColorado State University, USA

Leluo GuanUniversity of Alberta, Canada

Joshua GurtlerERRC, USDA-ARS, USA

Yong D. HangCornell University, USA

Divya JaroniOklahoma State University, USA

Weihong Jiang Shanghai Institute for Biol. Sciences, P.R. China

Michael JohnsonUniversity of Arkansas, USA

Timothy KellyEast Carolina University, USA

William R. KenealyMascoma Corporation, USA

Hae-Yeong Kim Kyung Hee University, South Korea

W.K. KimUniversity of Manitoba, Canada

M.B. KirkhamKansas State University, USA

Todd KostmanUniversity of Wisconsin, Oshkosh, USA

Y.M. Kwon University of Arkansas, USA

Maria Luz Sanz MuriasInstituto de Quimica Organic General, Spain

Melanie R. MormileMissouri University of Science and Tech., USA

Rama NannapaneniMississippi State University, USA

Jack A. Neal, Jr.University of Houston, USA

Benedict OkekeAuburn University at Montgomery, USA

John PattersonPurdue University, USA

Toni Poole FFSRU, USDA-ARS, USA

Marcos RostagnoLBRU, USDA-ARS, USA

Roni ShapiraHebrew University of Jerusalem, Israel

Kalidas ShettyUniversity of Massachusetts, USA

EDITORIAL BOARD

80 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

EDITOR-IN-CHIEFSteven C. RickeUniversity of Arkansas, USA

EDITORSTodd R. CallawayFFSRU, USADA-ARS, USA

Cesar CompadreUniversity of Arkansas for Medical Sciences, USA

Philip G. CrandallUniversity of Arkansas, USA

MANAGING EDITOR

Dave EdmarkFayetteville, AR

LAYOUT EDITOREllen J. Van LooGhent, Belgium

TECHNICAL EDITORJessica C. ShabaturaEureka Springs Arkansas, USA

ONLINE EDITION EDITORC.S. ShabaturaEureka Springs Arkansas, USA

ABOUT THIS PUBLICATION

Agriculture, Food & Analytical Bacteriology (ISSN

2159-8967) is published quarterly, beginning with

this inaugural issue.

Instructions for Authors may be obtained at the

back of this issue, or online via our website at

www.afabjournal.com

Manuscripts: All correspondence regarding pend-

ing manuscripts should be addressed Ellen Van Loo,

Managing Editor, Agriculture, Food & Analytical

Bacteriology: [email protected]

Information for Potential Editors: If you are interested

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tact Editor-in-chef, Steven Ricke, Agriculture, Food &

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Reprint Permission: Correspondence regarding re-

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EDITORIAL STAFF

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 81

TABLE OF CONTENTS

Influence on Growth Conditions and Sugar Substrate on Sugar Phosphorylation Activity in Acetogenic BacteriaW. Jiang, R.S. Pinder, and J.A. Patterson

94

A Membrane Filtration Method for Determining Minimum Inhibitory Concentrations of Essential OilsS. J. Pendleton, R. Story, C. A. O’Bryan, P. G. Crandall, S. C. Ricke, and L. Goodridge

88

Effect of Fertilization on Phytase and Acid Phosphatase Activities in Wheat and Barley Cultivated in Bulgaria

V. I. Chalova, I. Manolov, M. Nikolova, and L. Koleva

103

Transfer of Tylosin Resistance Between Enterococcus spp. During Continuous-Flow Culture of Feral or Domestic Porcine Gut Microbes N. Ramlachan, R.C. Anderson, K. Andrews, R.B. Harvey, and D.J. Nisbet

111

Sugar Recovery from the Pretreatment/Enzymatic Hydrolysis of High and Low Specific Gravity Poplar ClonesA. C. Djioleu, A. Arora, E. M. Martin, J. A. Smith, M. H. Pelkki, and D. J. Carrier

121

Culture dependent molecular analysis of bacterial community of Hazaribagh tannery exposed area in BangladeshA. A. Maruf, M. M. Moosa, S. M. M. Rashid, H. Khan, and S. Yeasmin

132

ARTICLES

Evaluation of an Experimental Sodium Chlorate Product, With and WithoutNitroethane, on Salmonella in Cull Dairy CattleN. A. Krueger, T. S. Edrington, R. L. Farrow, R. Hagevoort, R. C. Anderson, G. H. Loneragan,

T. R. Callaway, and D. J. Nisbet

82

BRIEF COMMUNICATIONS

Instructions for Authors149

Introduction to Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

Impact of By-product Feedstuffs on Escherichia coli O157:H7 and Salmonella Typhimurium in Pure and Mixed Ruminal and Fecal Culture in VitroT. R. Callaway, S. Block, K. J. Genovese, R. C. Anderson, R. B. Harvey, and D. J. Nisbet

139

82 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

www.afabjournal.comCopyright © 2012

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Ruminant animals are natural reservoirs for Salmonella. These bacteria can reduce nitrate to nitrite

through the membrane bound enzyme nitrate reductase which also has the ability to reduce chlorate to

the cytotoxic end-product chlorite. An experimental product containing sodium chlorate (ECP) has been

investigated in recent years as a pre-harvest food safety strategy to reduce Salmonella. The addition of ni-

troethane has been shown to enhance the effectiveness of ECP. The objective of this research was to deter-

mine if feeding ECP, with and without nitroethane, is effective in reducing naturally occurring populations

of Salmonella in cull dairy cattle on a commercial dairy prior to slaughter. Twelve cull dairy cows, dosed

for two consecutive days with either 140 mg of ECP containing 30% sodium chlorate /kg BW/d or with 70

mg of the ECP plus 160 mg nitroethane /kg BW/d, were sampled 48 h post initial dose at 12 h intervals for

Salmonella via fecal samples. Upon completion of the 48 h sampling animals were necropsied and gastro

intestinal tissue and luminal content samples taken for bacterial enumeration. The data presented herein

support the use of chlorate as a pre-harvest intervention strategy for reducing Salmonella in cull dairy cows

prior to entering the food chain can serve as an effective means of reducing these bacteria.

Keywords: Sodium Chlorate product, Salmonella, nitroethane, cull dairy cattle

InTRoduCTIon

According to the Centers for Disease Control and

Correspondence:Thomas S. [email protected]: +1 979-260-3757 Fax: +1 979-260-9332

Prevention, approximately 48 million people get sick

each year from foodborne diseases in the United

States (CDC 2010). The bovine gastrointestinal tract

is a well recognized reservoir for bacterial patho-

gens like Escherichia coli O157:H7, Salmonella and

Campylobacter. In the United States these bacterial

BRIEF COMMUNICATIONS

Evaluation of an Experimental Sodium Chlorate Product, With and Without Nitroethane, on Salmonella in Cull Dairy Cattle †

N. A. Krueger1, T. S. Edrington1, R. L. Farrow1, R. Hagevoort2, R. C. Anderson1, G. H. Loneragan3, T. R. Callaway1 and D. J. Nisbet1

1 United States Department of Agriculture, Agriculture Research Service, Southern Plains Agriculture Research Center, Food and Feed Safety Research Unit, 2881 F&B Road, College Station, TX 77845 USA.

2 Agriculture Experiment Station, New Mexico State University, Clovis, NM 88101-9998 USA.3Department of Animal and Food Sciences, Texas Tech University, PO Box 42141, Lubbock, Texas 79409-2141 USA.

† Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warrenty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.

Agric. Food Anal. Bacteriol. 2:82-87, 2012

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 83

pathogens are responsible for more than 3.5 million

human infections annually at an estimated annual

cost of more than $3.5 billion a year (ERS/USDA,

2009). According to Wells and others (Wells et al.,

2001), as many as 66% of the cull dairy cows have

detectable amounts of Salmonella shedding, and

these cull cows contribute substantially to the beef

supply, especially ground beef. Thus, pre-harvest

intervention strategies that reduce the shedding of

food-borne pathogens in cull dairy cattle are essen-

tial to reducing the amount of pathogenic bacteria

entering slaughter facilities and contributing to con-

tamination of food products and potentially human

infections.

Certain bacteria, such as Salmonella have the abil-

ity to reduce nitrate to nitrite through the intracel-

lular enzymes nitrate reductases (NarA and NarZ)

(Alaboudi 1982; Moreno-Vivia et al., 1999). It has

been suggested that the NarA enzyme, which is

expressed under anaerobic conditions, is primary a

contributor to the reduction of nitrate to nitrite (An-

derson et al., 2006a). However, the NarZ enzyme can

account for approximately 10% the nitrate reduc-

tase activity (Anderson et al., 2006a). Furthermore,

these reductase enzymes have the ability to also re-

duce chlorate to the cytotoxic end-product chlorite

(Stewart 1988, Fox et al., 2005 and Moreno-Vivia et

al., 1999). In recent years, chlorate supplementation

has been investigated as a pre-harvest food safety

strategy to reduce Salmonella and E. coli O157:H7

in vitro and in food producing animals (Anderson et

al., 2000). Research also has demonstrated effects

against Salmonella in swine and poultry (Anderson

et al., 2001; Anderson et al., 2004; Burkey et al., 2004)

but to date the effect on Salmonella in cattle has not

been evaluated. Research has shown that the addi-

tion of short chained nitro compounds like nitroeth-

ane can enhance the ability of sodium chlorate to

reduce Salmonella as much as ten-fold in vitro and in

vivo (Anderson et al., 2006a & 2006b). The objectives

of the current research was to determine if feeding

an experimental sodium chlorate product, with and

without nitroethane, is effective in reducing popula-

tions of Salmonella, in cull dairy cattle on a commer-

cial dairy prior to slaughter.

MATeRIAlS And MeThodS

All cattle were obtained from a conventional com-

mercial dairy in the Southern High Plains of the Unit-

ed States and were cared for according to guidelines

pre-approved for by the Southern Plains Agriculture

Research Center’s Animal Care and Use Committee

(ACUC no. 2010005). Dairy cows that were sent to

the hospital pen per the dairy’s standard operating

procedures were prescreened for Salmonella. The

dairy’s hospital pens are used to house animals that

were truly “sick” animals as well as animals that were

needing to be separated from the rest of the herd,

due to significant loss in milk production, laminitis,

mobility issues, etc., which classified them as poten-

tial candidates to be culled from the herd per the

discretion of the herd manager and standard operat-

ing procedures. Cows were restrained in self-locking

head stalls and approximately 30 g of fecal mate-

rial was obtained via rectal palpation. Fecal samples

were shipped on ice to the Food and Feed Safety

Research Unit in College Station, TX, USA (FFSRU)

for culture of Salmonella the following day. Five days

post-pre screen sampling, animals confirmed as Sal-

monella positive were enrolled. Twelve lactating Hol-

stein dairy cows (average BW 545 kg) testing positive

for Salmonella were purchased from the dairy and six

animals were randomly assigned to each treatment

(chlorate or chlorate + nitroethane). All experimental

animals remained on the dairy and were housed in

a pen separate from the rest of the herd; otherwise

all feeding and management schemes were as nor-

mal for the dairy. As all experimental animals were

housed together in the same pen, cross contamina-

tion among animals was a possibility. However, in

the production setting, culled animals are exposed

to other potential carriers throughout their time up

to slaughter. Therefore we felt that co-mingling in a

pen would give a more “real-world” test of the ex-

perimental treatment.

Salmonella positive animals received either 140

mg of an experimental sodium chlorate product

(ECP)/kg BW/d or 70mg ECP plus 160 mg nitroeth-

ane/kg BW/d. Based on previous research (Anderson

et al., 2006a), a sub optimal dose of ECP with added

84 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

nitroethane enhanced the bactericidal effects of the

ECP against Salmonella. The ECP was a proprietary

product provided by EKA Chemicals Inc. (Marrietta,

GA) and contained 30% sodium chlorate by weight.

Nitroethane was administered in the form of nitro-

ethane salt (Majak et al., 1986). Treatments were ad-

ministered 4 times at 12 h intervals via stomach tube.

Animals were restrained in headstalls in the A.M. ac-

cording to the dairy’s standard operation, and for the

P.M. dose animals were moved to a chute for proper

head restraint. Treatments were mixed with approxi-

mately 100 mL of water just prior to dosing to allow

for adequate volume and fluidity for passage through

the stomach tube. Each treatment was then followed

with approximately 100 mL of water, prior to removal

of the stomach tube, to remove any treatment that

might have adhered to the stomach tube during dos-

ing. Fecal grab samples were collected immediately

prior to first dosing and subsequently every 12 h for

the next 48 h post initial dosing. Following the last

fecal collection animals were euthanized accord-

ing to the American Veterinary Medical Association

guidelines on euthanasia. Luminal contents and tis-

sues from the rumen, small intestine, cecum, spiral

colon and rectum were aseptically collected upon

necropsy. All samples were shipped daily on ice to

the FFSRU for quantitative and qualitative bacterial

culture.

Fecal and luminal contents were processed upon

arrival for qualitative and quantitative analysis of Sal-

monella (Edrington et al., 2009). Serogrouping of Sal-

monella-positive samples was conducted using slide

agglutination with Salmonella antiserum (Becton,

Dickinson, Sparks, ND). Sample Salmonella popu-

lations were quantified by direct plating of the TSB

phosphate ⁄ sample mixture (10 g sample + 90 mL

TSB; prior to enrichment) onto XLD agar (Oxoid, LTD,

Basingstoke, Hampshire, England) using a commer-

cially available spiral plater (Spiral Biotech Autoplate

4000; Advanced Instruments, Inc., Norwood, MA),

with a limit of detection of 1.99X102 CFU/g. Plates

were incubated overnight at 37°C. Colonies were

counted and concentrations calculated. Morphologi-

cally typical (black) colonies were confirmed as Sal-

Table 1. Effect of ECP on Salmonella presence in enriched and direct plated feces over time and tissues at necropsy. A number represents the results of the direct plating (quantitative culture) expressed as cfu (log10/g feces), whereas a positive or negative symbol indicates a negative result from spiral plating and either a positive or negative result following enrichment and culture of the sample (qualitative culture).

Animal ID

10310 11875 11271 4705 4630 5773

pre-screen, d -5 + + + + 4.96 6.20

Time, (h)0 + + + + + +12 + + + - + +24 - - - - - +36 - - - + - +48 - + - - + +

Tissue site

Cecum - - - - - +Spiral colon - - - - - +Small intestine + - - - 4.88 -Rumen - - - - - -Rectum - - - - - +

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 85

monella as described above and counted.

ReSulTS And dISCuSSIon

The scope of this experiment was to evaluate the

effects of ECP with and without nitroethane on fe-

cal shedding of Salmonella in cull dairy cows. The

experiment was performed on the farm to ensure

that daily farm practices and feeding regimes were

employed in an effort to accurately simulate on-

farm application of the ECP and ECP + nitroethane

products. Two animals in the ECP treatment group

were shedding Salmonella at high concentrations at

the time of pre-screening (Table 1). Figure 1 dem-

onstrates the ability of ECP to effectively reduce (5

log10 cfu/g feces) Salmonella fecal shedding concen-

tration in animals colonized with high concentrations

of Salmonella. The remaining animals were positive

for Salmonella following enrichment at pre-screen-

ing (Tables 1 and 2). Forty-eight hours post initial

treatment with ECP all animals were negative for

Salmonella via spiral plating and only 50% of the ani-

mals had any detectable amount of Salmonella from

enriched samples (Table 1). The ECP + nitroethane

treatment resulted in all animals testing negative

for Salmonella via spiral plating and only 33% of the

animals had any detectable amount of Salmonella

from enriched samples (Table 2). Luminal contents

yielded no detectable Salmonella via direct plating

for all animals regardless of treatment except animal

4630, which had 4.88 log10 CFU’s present in the small

intestine. All other sites for this animal were nega-

tive via spiral plating. One colony from each positive

sample was serogrouped, of which 32, 25, 18, 17, 5,

and 3 % were K, E1, C1, poly A-I, C2, and I respectively.

We recognize that there are potential criticisms

associated with the current research. The lack of

control animals in this study can beg the question as

to whether there was an effect due to the ECP or a

natural response in cattle that have been shedding

for several days. As with any experimental research

product, federal approval must be obtained before

animals can enter the food chain or rendering pro-

cess, and since ECP is still awaiting federal approval

all animals were required to be purchased from the

dairy. Upon termination of the study, animals were

composted on the dairy to prevent entrance to the

food or rendering chain. Additionally, due to the as-

sociated cost and welfare issues we did not utilize

control animals. The authors recognize this is a weak-

ness of the study but did not have funding required

to euthanize six perfectly healthy animals that could

otherwise be sold by the dairy. By selecting animals

that were persistently and consistently shedding Sal-

monella for five days prior to initial dosing with the

0

1

2

3

4

5

6

0 12 24 36 48

Log

10

CF

U g

-1

Time, h

Cow 4630

Cow 5773

Figure 1. Effect of ECP supplementation on Salmonella concentrations in feces over time through direct plating.

86 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

experimental treatments, there was a high likelihood

that the response seen once experimental treat-

ments were imposed would be due to the effects of

the experimental treatments. Granted, in hindsight it

would have been beneficial to have control animals

that provided fecal samples even if they were not

necropsied. However, extensive research by the au-

thors has repeatedly demonstrated the sporadic na-

ture of fecal shedding of Salmonella in dairy cattle.

Therefore we felt that luminal populations through-

out the digestive tract would be a better indication

of the actual “Salmonella status” of the animal and

be a better determinant of the effectiveness of the

treatments examined in this research. While we rec-

ognize that further experimentation is needed, the

data presented herein while not conclusive does

support the idea of using chlorate as a pre-harvest

intervention in cull dairy cattle.

ConCluSIonS

Administration of ECP on farm immediately fol-

lowing the decision to cull and prior to shipping

should allow adequate time for the chlorate to exert

its lethal effect on Salmonella prior to the animal en-

tering the abattoir. While the ECP did not kill 100%

of the cultured Salmonella, it did appear to reduce

populations in the high shedders to levels that are

effectively controlled by modern processing inter-

vention strategies. It is unknown why the nitroethane

did not enhance the bactericidal effect of chlorate

in this research as has been observed previously.

Further investigation is needed and research should

examine the effectiveness of on-farm ECP adminis-

tration by following cull animals through the harvest

process.

ACknowledgeMenTS

This project was funded in part, by beef and veal

producers and importers through their $1-per-head

checkoff and was produced for the Cattlemen’s Beef

Board and state beef councils by the National Cat-

tlemen’s Beef Association.

RefeRenCeS

Alaboudi, A. R. 1982. Microbiological studies of ni-

trate and nitrite reduction in the ovine rumen.

Ph.D. dissertation. University of Saskatchewan, SK,

Canada.

Table 2. Effect of ECP with added nitroethane on Salmonella presence in feces over time and for luminal tissues at necropsy following enrichment and culture (qualitative culture).

Animal ID16487 86 7986 4907 6415 5538

pre-screen, d -5 + + + + + +Time, (h)

0 + - + + + +12 - - - + - -24 - + - - - -36 + - + - + +48 - - + - - +

Tissue site

Cecum - - - - - -Spiral colon - - - - - -Small intestine - - + - - -Rumen + - - + + +

Rectum + - + + - -

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 87

Anderson, R. C., S. A. Buckley, L. F. Kubena, L. H.

Stanker, R. B. Harvey, and D. J. Nisbet. 2000. Bacte-

ricidal effect of sodium chlorate on Escherichia coli

O157:H7 and Salmonella typhimurium DT104 in ru-

men contents in vitro. J. Food Prot. 63:1038-1042.

Anderson, R. C., S. A. Buckley, T. R. Callaway, K. J.

Genovese, L. F. Kubena, R. B. Harvey, and D. J. Nis-

bet. 2001. Effect of sodium chlorate on Salmonella

Typhimurium concentrations in the weaned pig

gut. J. Food Prot. 64:255-258.

Anderson, R. C., M. E. Hume, K. J. Genovese, T. R.

Callaway, Y. S. Jung, T. S. Edrington, T. L. Poole, R. B.

Harvey, K. M. Bishoff, and D. J. Nisbet. 2004. Effect

of drinking-water administration of experimental

chlorate ion preparations on Salmonella enterica

serovar Typhimurium colonization in weaned and

finished pigs. Vet. Res. Comm. 28:179-189.

Anderson, R. C., Y. S. Jung, K. J. Genovese, J. L.

McReynolds, T. R. Callaway, T. S. Edrington, R. B.

Harvey, and D. J. Nisbet. 2006a. Low level nitrate

or nitroethane preconditioning enhances the bac-

tericidal effect of suboptimal experimental chlo-

rate treatment against Escherichia coli and Sal-

monella Typhimurium but not Campylobacter in

swine. Foodborne Path. Dis. 3:461-465.

Anderson, R. C., Y. S. Jung, C. E. Oliver, S. M. Hor-

rocks, K. J. Genovese, R. B. Harvey, T. R. Callaway,

T. S. Edrington, and D. J. Nisbet. 2006b. Effects of

nitrate or nitro supplementation on Salmonella en-

teric serovar Typhimurium and Escherichia coli in

swine feces. J. Food Prot. 70:308-315.

Burkey, T. E., S. S. Dritz, J. C. Nietfeld, B. J. John-

son, J. E. Minton. 2004. Effect of dietary mannano-

ligosaccharide and sodium chlorate on the growth

performance, acute-phase response, and bacterial

shedding of weaned pigs challenged with Salmo-

nella enterica serotype Typhimurium. J. Anim. Sci.

82:397-404.

Center for Disease Control and Prevention. 2010.

CDC Reports 1 in 6 Get Sick from Foodborne Ill-

nesses Each Year. http://cdc.gov/media/press-

rel/2010/r101215.html, accessed October 4, 2011.

Edrington, T. S., B. H. Carter, T. H. Friend, G. R. Ha-

gevoort, T. L. Poole, T. R. Callaway, R. C. Anderson,

D. J. Nisbet. 2009. Influence of sprinklers, used to

alleviate heat stress, on faecal shedding of E. coli

O157:H7 and Salmonella and antimicrobial suscep-

tibility of Salmonella and Enterococcus in lactating

dairy cattle. Lett Appl Microbiol. 48:738–743.

ERS/USDA data foodborne illness cost calculator.

Available at: http://www.ers.usda.gov/data/food-

borneillness. Accessed 23 June 2009.

Fox, J. T., R. C. Anderson, G. E. Carstens, R. K. Miller,

Y. S. Jung, J. L. McReynolds, T. R. Callaway, T. S.

Edrington, and D. J. Nisbet. 2005. Effect of nitrate

adaption on the bactericidal activity of an experi-

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Majak, W., K.-J. Cheng, J. W. Hall. 1986. Enhanced

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88 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

www.afabjournal.comCopyright © 2012

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Minimum inhibitory concentrations (MICs) of essential oils are commonly determined via broth dilution

method. These methods very rarely include a neutralization step, due to the complex nature of essential

oils and thus difficulties in finding an adequate neutralizer, which leads to the potential for continued action

of the oil on the bacteria during growth analysis. This study was devised to evaluate filtration as a means

to remove an essential oil, cold pressed terpeneless Valencia orange oil, from several species of bacteria,

and thus prevent further antibacterial action by the oil. A ninety-six well microtiter plate method was used,

followed by transfer of well contents to a ninety-six well filter plate. The contents of the filter plate were

washed twice to separate the oil from the bacteria. The bacteria were resuspended in a medium contain-

ing a growth indicator and transferred to a sterile 96 well microtiter plate to determine MICs. Escherichia

coli O157:H7 was inhibited at a concentration of 0.5±0.0%, Listeria monocytogenes at 0.5±0.0%, Staphy-

lococcus aureus at 0.31±0.13%, Salmonella Typhimurium at 0.31±0.13%, Shigella sonnei at 0.75±0.29%,

Yersinia enterocolitica at 0.31±0.13%, Enterococcus faecalis at 0.63±0.25%, Bacillus cereus at 0.44±0.13%,

and Pseudomonas aeruginosa exhibited complete resistance to the oil.

Keywords: Escherichia coli, Salmonella, Shigella, Yersinia, Enterococcus, Bacillus, Pseudomonas,

minimal inhibitory concentration, essential oil, membrane filtration

InTRoduCTIon

The main methods for determining minimum in-

hibitory concentrations (MICs) and minimum bacte-

ricidal concentrations (MBCs) of essential oils and

Correspondence: P. G. Crandall, [email protected] Tel: +1-479-575-7686

essential oil components are broth dilution, agar

diffusion, and disc diffusion (Fernandez-Lopez et al.,

2005; Friedly et al., 2009; Kim et al., 1995a,b; Kubo

et al., 2004; Nannapaneni et al., 2008; Tassou et al.,

2000). These methods for determining MICs or MBCs

do not commonly employ a neutralization step.

However, if no neutralization step is used when con-

ducting antimicrobial studies on surfaces or in food

BRIEF COMMUNICATIONA Membrane Filtration Method for Determining

Minimum Inhibitory Concentrations of Essential Oils

S. J. Pendleton1,3, R. Story1, C. A. O’Bryan1, P. G. Crandall1, S. C. Ricke1, L. Goodridge2

1 Center for Food Safety-IFSE and Food Science Department, 2650 Young Ave., University of Arkansas, Fayetteville, AR 72704

2Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523-11713Current address: Food Science and Technology, University of Tennessee, Knoxville, Tennessee 37996

Agric. Food Anal. Bacteriol. 2:88-93, 2012

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 89

matrices, the compounds being tested are still act-

ing on the bacteria when initial dilutions are made. In

order to determine the real effect of an antimicrobial

on a bacterium, neutralizers are frequently used to

inactivate the antimicrobial compound (Atwal et al.,

2010; Dey and Engley, 1983; Meers and Churcher,

1974). Essential oils are complex mixtures of differ-

ent compounds (Burt et al., 2005; Kubo et al., 2004;

O’Bryan et al., 2008), and currently used neutralizing

broths may not be suitable for use with these essen-

tial oils.

Meers and Churcher (1974) and Prince et al. (1975),

investigated the use of membrane filters to remove

antimicrobial compounds. Meers and Churcher

(1974) used a membrane to filter volumes of treated

cells in order to separate out the cells from antimicro-

bial drug treatments. The filters were washed, then

placed on agar, and incubated for 24 hours. Plates

were then counted to determine viable cells. Prince

et al. (1975) used a different approach. Cells were

first placed on the membrane and then exposed

to a disinfectant treatment for two and half or eight

minutes. The disinfectant was then filtered away and

the membrane was washed. The membrane was

then transferred to agar and incubated for 24 to 48

hours, after which counts were made to determine

antibacterial activity. Both techniques found that the

antimicrobial compounds were able to be effectively

removed from the cells via membrane filtration.

Membrane filtration offers the ability to remove

antibacterial compounds without the need for neu-

tralizers, especially when an adequate neutralizer is

not available (1). This aspect of membrane filtration

indicates its promise for the use of studying essential

oil antimicrobial activity. Therefore, the aim of this

study was to evaluate the efficacy of membrane fil-

tration in the determination of minimum inhibitory

concentrations for cold pressed terpeneless Valen-

cia orange oil against several species of bacteria.

MATeRIAlS And MeThodS

Essential oil

Commercially available cold pressed terpeneless

Valencia orange oil was obtained from Firmenich

(Lakeland, FL). Composition of this oil can be found

in Nannapaneni et al. (2009). The initial oil suspen-

sion was made by adding 0.2 mL of cold pressed ter-

peneless Valencia orange oil to 10 ml of tryptic soy

broth with 0.5% yeast extract (TSBYE) and 50 µL of

Tween 20 (Griffin et al., 2000; Kim et al., 1995a,b; Lin

et al., 2010) in a 10 ml centrifuge tube, giving 2% oil

and 0.5% (v/v) Tween 20 concentration.

Bacterial strains and growth

Nine bacterial strains from the culture collection

of the University of Arkansas Center for Food Safety

were used for this study: Escherichia coli O157:H7

(ATCC 43888), Listeria monocytogenes (USFDA),

Staphylococcus aureus (ATCC 25923), Salmonella

Typhimurium (ATCC 14028), Shigella sonnei (ATCC

25931), Yersinia enterocolitica (ATCC 23715), Entero-

coccus faecalis (ATCC 29212), Bacillus cereus (ATCC

11778), and Pseudomonas aeruginosa (ATCC 27853).

Cultures were grown in 10 mL of TSBYE in a 15 ml

centrifuge tube and incubated (GCA/Precision Scien-

tific, model 6M) at 37°C without shaking for 24 hours.

The cultures were then centrifuged (Damon,IEC Di-

vision, Needham, MA) at Relative Centrifugal Force

(RCF) = 4700 to 8500 x g for 10 min. The supernatant

was poured off and the pellet was resuspended in 10

ml of 20 mM PBS. A second wash was performed and

the final pellet was resuspended in TSBYE + 0.5%

Tween 20 and diluted to give resulting bacterial con-

centrations of approximately 106 CFU/mL.

Minimal inhibitory concentration

Two hundred µL of the oil suspension was added

into the first wells (column 1) of a sterile, 96 well tis-

sue culture plate. In the following wells (columns 2

through 6) 100 µL of TSBYE and 0.5% Tween 20 was

added to each well. With a multichannel pipette,

100 µL of oil in the first wells was transferred into the

second wells for a 1:2 dilution and followed by addi-

tional 1:2 dilutions. The final oil concentrations were

1%, 0.5, 0.25, 0.13, 0.063, and 0.03 (v/v). For nega-

90 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

tive controls, 100 µL of TSBYE and 0.5% Tween 20

was added to row G, to bring final volumes up to

200 µL. In triplicate, 100 µL of each 106 culture was

added to the various concentrations of oil (Figure

1.). An adhesive plate film (PlateMax: Axyseal Seal-

ing Film, pre-sterilized cat# PCR-SP) was placed over

the wells, and pressed down by hand. The plate was

incubated (New Brunswick Scientific, model # G-25)

for 24 hrs at 37°C with shaking at 200 rpm.

Filtration method

After incubation, 25 µL from each well was trans-

ferred into a matching sterile, 96 well filter plate

(0.2 um PVDF membrane, Corning, prod.# 3504). To

each well in the filter plate, 100 µL of TSBYE + 0.5%

Tween 20 was added. A sterile 96 well plate lid was

placed on the filter plate. The filter plate was then

placed on the collection plate (Corning, 0.5 ml, prod.

#3956) and centrifuged (Beckman TJ-6 centrifuge) at

RCF = 739 x g for 10 min. After centrifugation, 100

µL of TSBYE + 0.5% Tween 20 was added to each

well of the filter plate and then centrifuged again.

After centrifugation, 200 µL of TSBYE + 1% triphenyl

tetrazolium chloride (TTC) were added to each well

of the filter plate. With a multichannel pipette, the

contents of each well were gently mixed and trans-

ferred to a matching sterile 96 well plate. The plate

was statically incubated at 37°C for 48 hrs. The wells

were then visually checked for growth via develop-

ment of red pigment. Wells with the lowest oil con-

centration having no red pigment were considered

the MIC. The experiment was repeated four times.

ReSulTS And dISCuSSIon

The results of the minimum inhibitory concentra-

tion determinations can be found in Table 1. Cold

pressed terpeneless Valencia orange oil exacted

MICs of 0.5%, 0.5%, 0.31%, and 0.31% against E.

coli O157:H7, L. monocytogenes, S. aureus, and

S.Typhimurium, respectively. MICs of 0.75%, 0.31%,

0.63%, and 0.44% were exacted against S. sonnei,

Y. enterocolitica, E. faecalis, and B. cereus, respec-

tively. P. aeruginosa exhibited complete resistance

to the highest concentration of the oil.

Prior studies have evaluated the effects of cold

Figure 1. 96-well microtiter plate design for MICs with cold pressed terpeneless Valencia orange oil concentrations in percentages.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 91

pressed terpeneless Valencia orange oil against dif-

ferent bacteria using non-membrane filtration tech-

niques (Friedly et al., 2009; Nannapeneni et al., 2008;

O’Bryan et al., 2008). Friedly et al. (2009) examined

the effect of the essential oil on strains of L. mono-

cytogenes and Listeria innocua. It was determined

that the oil exhibited a MIC of 0.55% against both

L. monocytogenes and L. innocua after 18 hours of

incubation at 37°C. This result is very similar to the

result found in the current study, indicating that the

antibacterial inhibition of L. monocytogenes by the

oil does not continue after 18 hours of incubation at

37°C. O’Bryan et al. (2008) determined the antibac-

terial effect of the oil against strains of Salmonella,

including a strain of S. Typhimurium (Copenhagen).

Cold pressed terpeneless Valencia orange oil ex-

hibited little effect on the strain of S. Typhimurium

tested. The zone of inhibition determined by disc

diffusion was only 7.3±1.2 mm, which included the

6 mm paper disc. The results of the current study

do not agree with this lack of antibacterial activity

against S. Typhimurium. This could possibly be due

to differences between strains of Typhimurium, al-

though further research is needed to confirm this

hypothesis. Nannapaneni et al. (2008) examined the

ability of cold pressed terpeneless Valencia orange

oil to inhibit the growth of E. coli O157:H7 strains.

The oil, tested by disc diffusion, produced a zone of

inhibition against E. coli O157:H7 (ATCC 43888) of

11.5±0.7 mm. It is not stated in the study if the in-

hibition zone includes the paper disc, but even if it

does, the oil exhibited a much higher inhibition of E.

coli O157:H7 (ATCC 43888) than S. Typhimurium (Co-

penhagen). This result is contradicted by the current

study, in which both E. coli O157:H7 (ATCC 43888)

and S. Typhimurium were found to be inhibited at

the same concentration. The contrast in results could

be due to the difference in methods used to deter-

mine antibacterial susceptibility, as well as a differ-

ence in strains of S. Typhimurium. The current study

used a micro-broth dilution method, while the other

studies used disc diffusion methods. The activity of

cold pressed terpeneless Valencia orange oil possi-

bly could be increased in a broth medium, although

further study is needed to elicit an answer.

The responses of the other bacteria to cold

pressed terpeneless Valencia orange oil have not

been previously determined. Therefore, it is not pos-

sible to compare the results of this study to others. It

can be said that the current method was able to elicit

Table 1. Minimum inhibitory concentrations of cold pressed Valencia orange oil for 9 bacterial strains at 37°C.

Bacterial Strain MIC (%)

E. coli O157:H7 0.5

L. monocytogenes 0.5

S. aureus 0.31

S. Typhimurium 0.31

S. sonnei 0.75

Y. enterocolitica 0.31

E. faecalis 0.63

B. cereus 0.44

P. aeruginosa >10

92 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

MICs for all other bacteria tested, except P. aerugi-

nosa. The inability to determine an MIC for P. aeru-

ginosa is most likely due to its inherent resistance to

essential oil components (Cox and Markham, 2007).

Cox and Markham (2007), found that P. aeruginosa

NCTC 9027 and 6749 were tolerant of 6 of 8 essential

oil components. Only cinnamaldehyde and carvacrol

showed any inhibition of the bacteria. It was deter-

mined that these bacteria contained an active efflux

mechanism, which allows them to resist the antibac-

terial effects of many essential oil components.

ConCluSIonS

The current method tested was able to produce

reliable MICs, but was unable to indicate the need

for membrane filtration in the MIC determinations

of essential oils. The MICs determined in this study

were either similar to those found by traditional

methods, or lower than those found by traditional

methods. These results are contrary to that of the

original hypothesis: MICs should be lower in tradi-

tional methods due to residual inhibition by essen-

tial oils. Replication of these results, as well as side

by side comparison of methods is needed to confirm

this conclusion.

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94 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

www.afabjournal.comCopyright © 2012

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Four acetogenic bacteria isolates (designated as A2, A4, A10 and H3HH) were tested for phosphoenol-

pyruvate (PEP) and ATP-dependent phosphorylation of glucose and 2-deoxy-glucose. Although all organ-

isms had detectable phosphorylation activity, substantial variation existed in the rates of both PEP- and

ATP-dependent phosphorylation. Isolate A10 had the highest rate of PEP- dependent phosphorylation

(net rate of 11.62 nM per mg protein per min). Isolate A10 as well as isolates A2 and H3HH most likely

have a glucose phosphotransferase system (PTS). In contrast, isolate A4 had PEP-dependent glucose

phosphorylation rates very similar to control rates, suggesting the lack of PTS activity. These results were

confirmed by PEP dependent 2-deoxyglucose phosphorylation data. The rates of ATP-dependent glucose

phosphorylation were higher than PEP-dependent glucose dependent in all organisms surveyed. How-

ever, substantial variation existed for ATP-dependent glucose phosphorylation rates. The glucose PTS

of isolates A10 and H3HH were induced by the presence of extracellular glucose. Moreover, the specific

activity of the glucose PTS of both isolates increased as cultures progressed from the early log to late log

phase of growth. ATP- and PEP-dependent maltose and sucrose phosphorylation was detected in isolates

A10 and H3HH. Although activity was detected in both isolates (A10 and H3HH), the rate of activity varied

considerably, depending on the sugar and organism tested.

Keywords: Acetogenic bacteria, phosphoenolpyruvate (PEP), ATP-dependent phosphorylation,

glucose, 2-deoxy-glucose, rumen, acetate

InTRoduCTIon

The key to survival in a particular ecosystem (such

as the rumen) is the ability to adapt to changes of the

Correspondence: John Patterson [email protected]  Tel: +1 -765-494-4826 Fax: +1-765-494-9347

ecosystem. Uptake of nutrients is a determinant of

the success of bacteria in the natural environments

(Matin and Veldkamp, 1978). Therefore, the ability

of an organism to adapt its sugar transport systems

to the prevailing conditions of its environment is es-

sential for its survival. The second objective was to

determine the effects of growth conditions (i.e en-

Influence on Growth Conditions and Sugar Substrate on Sugar Phosphorylation Activity in Acetogenic Bacteria

W. Jiang1, 2, R.S. Pinder2 ,3, and J.A. Patterson2*

1Current address: Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences

2Department of Animal Sciences, Purdue University. West Lafayette, IN 479073Current address: 7855 South 600 East, Brownsburg, IN 46112

Agric. Food Anal. Bacteriol. 2:94-102, 2012

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 95

ergy source, stage of growth and presence of other

sugars) on the phosphorylating activity of acetogen-

ic bacteria. These PTS are capable of translocating

glucose, maltose and sucrose and are induced by

the presence of carbohydrates.

Because of the uniqueness and potential for utili-

zation of the Wood pathway (Ragsdale, 1991), much

of the attention focused on acetogens has concen-

trated on characteristics of this pathway. Conse-

quently, relatively little information is known about

other metabolic systems in these organisms. Al-

though acetogenic organisms are capable of utiliz-

ing sugars, the mechanisms responsible for uptake

and utilization of these compounds remain largely

unknown. Another motive for examining the sug-

ar transport system of acetogenic bacteria was to

explore the possibility that H2 utilization could be

regulated by the presence of sugars through the ac-

tivities of a PTS. Because bacterial PTS are known to

affect the metabolic activity of other energy systems

in bacterial cells, in part through the action of cAMP

and its receptor protein (CPR) on gene transcription

(Saier, 1991), our hypothesis was that some aceto-

gens do utilize the PTS system. Therefore, the first

objective for the research reported herein was to de-

termine the presence of PTS activity in acetogenic

ruminal isolates.

MATeRIAlS And MeThodS

Organisms, growth conditions and cell treatment

Several ruminal acetogenic bacteria were isolated

from dairy cattle and were characterized (Boccazzi

and Patterson, 2011; Pinder and Patterson, 2011).

Acetogenic medium (Boccazzi and Patterson, 2011)

was supplemented with sugars (glucose, maltose

or sucrose) as indicated in the results and discus-

sion section. Stock solutions of sugars were pre-

pared separately as anaerobic solutions (5% w/v),

autoclaved separately and aseptically added to the

acetogen medium once both the sugar solution and

acetogen medium had reached room temperature.

Acetogen medium (20 mL) was dispensed into 120

ml volume serum tubes and sealed with butyl rub-

ber stoppers. All of the gases used in the study were

passed through a copper furnace to remove residual

oxygen and unless mentioned otherwise consisted of

100% CO2.

Cells in 20 mL of acetogen medium were harvest-

ed by centrifugation (10,000 x g, 10 min, 4°C) washed

twice with NAKP buffer (50 mM sodium phosphate,

50 mM potassium phosphate, 5 mM MgCl2, and 1

mM dithiotreitol; pH 7.2), suspended in a toluene-

ethanol mixture (1:9) followed by vortexing for 1 min

and kept on ice before being used as described by

Martin and Russell (1986).

Phosphorylation assays

Cell samples were treated with 0.2 N NaOH (100°C,

15 min) prior to protein determination and protein

concentration was estimated with a Bio-Rad protein

assay (Richmond, CA) with bovine serum albumin

used as the protein standard. For the rate of sugar

phosphorylation measurement in toluene-treated

acetogenic cells the reaction mixture (1 mL) con-

tained NAKP buffer supplemented with 10 mM PEP

or 10 mM ATP and 0.1 mL of toluene-treated cells as

described by Martin and Russell (1986) as modified by

Jiang et al. (2012). The reaction for phosphorylation

was begun by the addition of the respective (1mM)

sugar containing either 0.2 µCi of D-[U-14C]-glucose,

2-deoxy-D-[U-14C]-glucose, [U-14C]-maltose or [U-14C]-

sucrose. ATP, PEP, [14C]-labeled and unlabeled glu-

cose, 2-deoxyglucose, maltose, and sucrose as well

as dithiohthreitol and BaBr2 were purchased com-

mercially (Sigma Chemical Co., St Louis, MO). After

incubation (39° C for 30 minutes) BaBr2 (10 mL of 30

mM in 90% ethanol) was combined with the reac-

tion mixture followed by further incubation (20 min-

utes on ice). Precipitated phosphorylation products

from the reaction mixture were retrieved with a 0.45

µm pore membrane filter (Millipore Corp., Bedford,

Mass), rinsed with 80% ethanol, air dried and counted

in a 1600-R liquid scintillation counter. Finally, influ-

ences of unlabeled glucose, maltose, or sucrose on

PEP- or ATP-dependent phosphorylation influences

96 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

were determined by adding each respective sugar

(10 mM) the reaction mixtures. Sugar phosphoryla-

tion by endogenous sources of PEP and ATP was as-

sessed in control assays without exogenous PEP or

ATP. All experiments were conducted is triplicates

and estimates of variation are presented as standard

deviations.

ReSulTS And dISCuSSIon

PEP- and ATP-dependent glucose phos-phorylation

ATP conservation is critical to anaerobic bacteria

because they lack of a complete TCA cycle severely

curtails the yield of ATP from glycolysis (Thauer et al.,

1977). PEP-dependent glucose phosphorylation was

detected in all four acetogenic bacteria tested (Ta-

ble 1). Among the organisms tested, isolate A10 had

the highest rate of PEP-dependent glucose phos-

phorylation (net rate of 11.33 nM of glucose phos-

phorylated per min) While isolate A4 had the lowest

net rate of PEP-dependent glucose phosphorylation

(0.15 nM of glucose phosphorylated per mg of cell

protein per min). These phosphorylation rates are

comparable to those of other ruminal bacteria (e.g

Prevotella ruminicola, Selenomonas ruminantium,

and Streptococcus bovis) surveyed by Martin and

Russell (1986, 1987). The rates of PEP-dependent

glucose phosphorylation determined with cells of

isolate A4 were so low that this organism most likely

does not possess a glucose PTS. In all the organisms

tested, the rate of ATP-dependant phosphorylation

was greater than that of PEP-dependent phosphory-

lation. This pattern is consistent to that observed

in nonacetogenic bacteria as well as acetogens (Ji-

ang et al., 2012; Martin and Russell, 1986; Moore and

Martin, 1991). Nevertheless, as observed with PEP-

dependent phosphorylation, isolate A4 did not pos-

sess relevant glucose transport systems dependent

on either PEP or ATP. Neither A2 or A4 grows vigor-

ously using glucose as the growth substrate, which

reinforces our conclusions.

The rates of 2-deoxyglucose phosphorylation

were slightly lower than those of glucose phosphory-

lation in all the acetogenic organisms tested (Table

1). In contrast, isolate A10 had a rate of PEP-depen-

dent 2-deoxyglucose phosphorylation approximate-

ly 20 fold greater than control, strongly suggesting

the presence of a PTS in this organism. The other

organism tested (isolate H3HH) had PEP-dependent

2-deoxyglucose phosphorylation rates that were be-

tween 10 and 20 times greater than ATP-dependent

2-deoxyglucose phosphorylation, suggesting that

these two organisms most likely contain a PTS as

well.

Table 1. Specific activities of glucose and 2-deoxyglucose phosphorylation with PEP or ATP as phosphoryl donors in acetogenic bacteria1

Glucose 2-deoxyglucose

acetogens none PEP ATP none PEP ATP

A2 1.38±0.10 2.18±0.08 20.45±0.99 ND2 ND ND

A4 1.05±0.18 1.20±0.13 3.27±0.11 ND ND ND

A10 0.29±0.02 11.62±0.06 14.01±0.52 0.31±0.03 11.14±0.32 0.35±0.01

H3HH 1.39±0.09 7.07±0.19 9.99±0.43 1.25±0.05 4.25±0.36 1.40±0.03

1Nanomoles of glucose or 2-deoxyglucose phosphorylated per milligram of protein per minute. Values are mean ± standard deviation (n=3).2ND=not determined.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 97

Effect of growth on glucose phosphory-lating activity of acetogenic cells

The specific activity of the glucose PTS in isolates

H3HH and A10 was not constant. Cells of isolate

H3HH and A10 grown on glucose had 9- and 4- fold

greater activity, respectively, then cells grown solely

on H2 + CO2 (Table 2). Conversely, the rate of ATP

dependant phosphorylation was unchaged regard-

less of whether the cells of either isolate A10 or H3HH

were grown on glucose or H2 + CO2. These results

suggest that ATP dependent glucose phosphoryla-

tion activity is constitutive while PEP-dependent glu-

cose phosphorylation is induced by the presence of

carbohydrates in cultures of acetogenic organisms.

Regulation of PTS activity by the presence of extra-

cellular carbohydrates is a well-documented phe-

nomenon (Saier, 1985). For example, Rephaeli and

Saier (1980) reported induction of several proteins of

the PTS (i.e. enzyme I, HPr, and the glucose specific

enzyme II) in response to the presence of extracel-

lular carbohydrates. Similar to glucose phosphory-

lation, the PEP-dependent 2-deoxy-glucose phos-

phorylation activity was induced in cells (of either

isolate A10 or H3HH) growing on glucose.

The specific activity of the PTS in cells grown sole-

ly on glucose was not constant (Table 3). The specific

activity of the PTS increased as the cultures of both

isolate A10 and H3HH progressed from early log to

late log growth phase. However, once the cultures

entered the stationary phase, glucose phosphoryla-

tion rates declined precipitously to levels lower than

those observed in cultures in early log growth phase.

A similar pattern was observed for ATP-dependent

phosphorylation. Feedback inhibition of the trans-

port systems by intracellular glucose-6-phosphate is

a plausible explanation for these events because in-

hibition of several enzyme II’s (the carbohydrate-spe-

cific permeases of the PTS) by glucose-6-phosphate

has been reported previously (Postma and Lengeler,

1985). During the early to mid log growth phase the

intracellular concentration of glucose-6-phosphate

would be relatively high because extracellular glu-

cose was in excess of the needs of the cells. However,

once the glucose supply was exhausted, the amount

of glucose entering the cells would decrease, caus-

ing the cells to enter the late log phase and begin to

slow down the growth rate. Additionally the intracel-

lular concentrations of glucose-6-phosphate would

decrease as well.

Effect of carbohydrate growth sub-strate on rates of glucose, maltose, and sucrose phosphorylation

Isolates H3HH and A10 are capable of grow-

Table 2. Effect of growth substrate on the specific activity PEP- and ATP- dependent phosphory-lation of glucose and 2-deoxyglucose by isolates H3HH and A101

Glucose 2-deoxyglucose

none PEP ATP none PEP ATP

H3HH

glucose 1.39±0.09 7.07±0.19 9.99±0.43 1.25±0.05 4.25±0.36 1.4±0.03

H2/CO2 0.48±0.05 0.83±0.16 9.20±0.26 0.25±0.02 0.42±0.07 0.42±0.1

A10

glucose 0.29±0.02 11.62±0.06 14.01±0.52 0.31±0.03 11.14±0.32 0.35±0.01

H2/CO2 0.96±0.08 3.57±0.13 13.05±0.23 0.56±0.1 2.86±0.02 0.71±0.061Nanomoles of glucose or 2-deoxyglucose phosphorylated per milligram of protein per minute.

Values are mean ± standard deviation (n=3).

98 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

ing on maltose and sucrose at rates similar to the

rate observed with growth on glucose ( Pinder and

Patterson, unpublished observations). Therefore,

we expanded our examination to encompass the

maltose and sucrose phosphorylation performed by

these isolates to gain addition information regard-

ing the sugar transports of these organisms. PEP-

and ATP- dependent phosphorylation of glucose,

maltose and sucrose was detected in both isolate

H3HH and isolate A10, regardless of the sugar (glu-

cose, maltose, or sucrose) used for growth (Tables

4 and 5). ATP- dependent glucose phosphorylation

was the highest phosphorylation activity detected

in both isolates, regardless of the growth substrate.

The rate of PEP- dependent glucose phosphoryla-

tion was higher than PEP- dependent maltose and

glucose phosphorylation in both isolates. Similarly,

S. ruminantium and S. bovis have higher rates of

Table 3. Effect of growth stage on the specific activity of PEP- and ATP-dependent phos-phorylation of glucose by isolates H3HH and A101

Growth period OD none PEP ATP

H3HH

Early log 0.5 0.73±.151 1.42±0.08 6.98±0.13

Mid log 1 0.56±0.06 3.30±0.05 9.91±0.5

Late log 1.5 1.09±0.16 10.33±0.74 20.32±0.55

Stationary 2.5 0.79±0.09 1.71±0.17 4.58±0.13

A10

Early log 0.3 0.57±0.02 3.18±0.13 6.55±0.33

Mid log 0.6 0.56±0.06 5.45±0.18 7.16±.09

Late log 1 0.46±0.11 15.58±0.06 16.94±.24

Stationary 1.2 0.66±0.08 0.91±0.04 2.91±0.04

1Nanomoles of glucose or 2-deoxyglucose phosphorylated per milligram of protein per minute. Values are mean ± standard deviation (n=3).

Table 4. Net specific activity of PEP- and ATP- dependent sugar phosphorylation of isolate H3HH growing on different growth substrates1

Glucose Maltose Sucrose

Growth

substrate

PEP ATP PEP ATP PEP ATP

glucose 2.98±0.01 28.14±0.36 1.43±0.09 0.56±0.34 0.19±0.1 ND2

maltose 1.77±.20 12.61±0.27 1.34±0.07 0.22±0.14 0.26±0.01 0.14±0.1

sucrose 1.63±0.02 23.8±0.36 1.57±0.3 0.3±.04 1.3±0.04 5.09±1.26

1 Nanomoles of glucose or 2-deoxyglucose phosphorylated per milligram of protein per minute. Values are mean ± standard deviation (n=3).2ND=No ATP-dependent phosphorylating activity was detected after subtracting the control values.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 99

PEP-dependent glucose phosphorylation as com-

pared to either maltose or sucrose phosphorylation

dependent on PEP (Martin and Russell, 1987, 1988).

ATP-dependent maltose phosphorylating activi-

ties were detected in both isolate A10 and H3HH.

However, the rates of these activates were so low in

both isolates (less than 1.3 fold greater than control

rates without exogenous PEP or ATP), that ATP- de-

pendent maltose phosphorylation probably does

not play a major role in maltose utilization by these

organisms. Neither glucokinases nor hexokinases

are able to phosphorylate maltose (Barman, 1969),

therefore it is not clear at present what enzymes

were responsible for the ATP-dependent maltose

phosphorylation activity detected in these isolates.

Possible candidates include maltose phosphorylase

or maltase. The two isolates (A10 and H3HH) dif-

fered in the rates of ATP-dependent maltose phos-

phorylation when grown on maltose or sucrose than

when grown on glucose, while isolate H3HH was op-

posite. Nevertheless, both isolates had similar rates

of PEP-dependent maltose phosphorylation, regard-

less of the sugar substrate for growth.

Sucrose activities (regardless of the phospho-

ryl donor) were highest when the organisms were

grown on sucrose, suggesting induction of a sucrose

PTS as well as sucrose kinase activity. Similar induc-

tion of PEP-dependent sucrose phosphorylation was

detected in Streptococcus bovis (Martin and Russell,

1987). The relative rates of ATP-and PEP-dependent

glucose phosphorylation of the two organisms sug-

gest that isolate H3HH phosphorylates sucrose pri-

marily through a sucrose kinase while isolate A10

uses primarily sucrose PTS. Nevertheless, the rates

of PEP-dependent sucrose phosphorylation in both

isolate A10 and H3HH were the lowest of all three

sugars tested.

Competition by other sugars

To determine whether uptake of glucose, maltose,

and sucrose was performed by the same or different

subsystems of the PTS in isolate A10 and H3HH, the

rate of phosphorylation of 14C-labeled sugar was de-

termined in the presence of a large (10mM) excess of

unlabeled sugar (Tables 6 and 7). The glucose and

maltose PEP-dependent phosphorylation activities

appear to originate from closely linked enzymes in

isolate H3HH, due to the relatively high amount of

reciprocal inhibition observed with these two sugars.

An excess of maltose inhibited 60% of 14C-glucose

phosphorylation, while and excess of glucose inhib-

ited 14C-maltose phosphorylation by 79%. A similar

pattern was observed in isolate A10, although the

degree of reciprocal inhibition was lesser. An excess

of maltose inhibited 51% of 14C-glucose phosphory-

lation, while an excess of glucose only inhibited 21%

of 14C-maltose phosphorylation. Furthermore, an

Table 5. Net specific activity of PEP- and ATP-dependent sugar phosphorylation of isolate A10 growing on different growth substrates1

Glucose Maltose Sucrose

Growth substrate PEP ATP PEP ATP PEP ATP

glucose 7.19±0.641 13.1±0.46 1.31±0.13 0.15±0.09 0.44±0.01 0.04±0.01

maltose 4.21±0.77 11.93±0.46 1.85±0.86 0.92±0.55 0.09±0.01 0.17±0.14

sucrose 7.59±0.47 8.56±0.52 1.95±0.02 0.64±0.03 1.12±0.03 0.34±0.03

1 Nanomoles of glucose or 2-deoxyglucose phosphorylated per milligram of protein per minute. Values are mean ± standard deviation (n=3).

100 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

excess of maltose only inhibited 14C-maltose phos-

phorylation by 44% in isolate A10 (compared to 94%

inhibition in isolate H3HH) which suggests that the

capacity for uptake and phosphorylation of maltose

is greater in isolate A10 than isolate H3HH. Recip-

rocal inhibition of PEP-dependent phosphorylation

was observed with combinations of glucose and

sucrose as well as maltose and sucrose in both iso-

lates. However, in isolate A10 the reciprocal inhi-

bition between maltose and sucrose was relatively

low (12 to 51% inhibition). Because the degree of

inhibition between the three sugars tested was not

always near the theoretical dilution (91% inhibition),

it appears that more than one subsystem (i.e. the

Table 6. Inhibition by unlabeled sugars of PEP- and ATP- dependent [14C]-sugar phosphoryla-tion in isolate H3HH

% inhibition of phosphorylation1

Glucose Maltose Sucrose

Radiolabeled sugar2 PEP ATP PEP ATP PEP ATP

glucose 87.1 72.6 60.5 NA3 39.1 NA

maltose 79 16.3 94.1 96 75.2 93.4

sucrose 59.1 79.2 51 85.3 84.3 NA

1Nanomoles of glucose or 2-deoxyglucose phosphorylated per milligram of protein per minute.% Inhibition was calculated as ((PTS activity with 10 mM unlabeled sugar/PTS activity without excess unlabeled sugar) x 100).2Cell were grown in excess of the same unlabeled sugar.3NA = No activity, experimental values were not greater than control values.

Table 7. Inhibition by unlabeled sugars of PEP- and ATP-dependent [14C]-sugar phosphorylation in isolate A10

% inhibition of phosphorylation1

Glucose Maltose Sucrose

Radiolabeled sugar2 PEP ATP PEP ATP PEP ATP

glucose 87.5 87.5 51.6 13.4 50.2 3.9

maltose 21.1 69.6 44.3 29.3 11.9 NA3

sucrose 24.1 73.5 29.5 35.3 50.9 79.41Nanomoles of glucose or 2-deoxyglucose phosphorylated per milligram of protein per minute.% Inhibition was calculated as ((PTS activity with 10 mM unlabeled sugar/PTS activity without excess unla-beled sugar) x 100).2Cell were grown in excess of the same unlabeled sugar.3NA = No activity, experimental values were not greater than control values.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 101

carbohydrate specific enzyme II and III) of the PTS

transports glucose, maltose and sucrose in the iso-

lates. Transport of glucose, maltose and sucrose by

different carbohydrate specific enzymes of the PTS

has been reported in other bacteria such as E. coli

(Meadow et al., 1990).

A much different picture emerged with ATP-de-

pendent phosphorylation of glucose, maltose and

sucrose in both isolate A10 and H3HH. Glucose

and maltose are phosphorylated by different ATP-

dependent systems in isolate H3HH. However, the

same relationship is less clear in isolate A10. Malt-

ose inhibited glucose phosphorylation by only 13%

while glucose inhibited maltose phosphorylation by

70% in isolate A10. This type if inhibition suggested

a feedback-type of inhibition by glucose upon ATP-

dependent maltose phosphorylation. A similar pat-

tern of feedback inhibition by glucose on sucrose

phosphorylation was observed in isolate A10.

Interestingly, an excess of unlabeled sucrose did

not reduce the rate of ATP-dependent 14C-sucrose

phosphorylation in isolate H3HH. Maltose and su-

crose are phosphorylated by the same enzyme sys-

tem in isolate H3HH in light of the relatively strong

reciprocal inhibition displayed between these two

sugars. The relationship between maltose and su-

crose ATP-dependent phosphorylation is less clear

in isolate A10. Sucrose did not inhibit maltose phos-

phorylation while maltose only achieved 29% inhibi-

tion on 14C-maltose phosphorylation and 35% inhibi-

tion on 14C-sucrose phosphorylation.

ConCluSIon

The constitutive nature of the ATP-dependent

glucose phosphorylating activities suggests that

this system is used as the initial system for uptake

of glucose in to the cell, and that the PTS serves as

a secondary system, in the same fashion as the am-

monia uptake systems of glutamate dehydrogenase:

glutamine synthetase. Further experiments will be

needed to determine the minimum concentration of

sugar needed to induce the PTS in these bacteria as

well as the mechanism how glucose uptake repress-

es activity of the Wood pathway.

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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 103

www.afabjournal.comCopyright © 2012

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Phytase and acid phosphatases in wheat and barley are the major enzymes which catalyze the release of

orthophosphate from phosphorylated substrates. Their activities may be influenced by numerous factors

including variety differences, growth conditions, and fertilization. The purpose of this study was to evaluate

the effect of nitrogen (N), phosphorus (P), and potassium (K) fertilization on phytase and acid phosphatase

activities in wheat and barley varieties which are developed and cultivated in Bulgaria. A randomized block

design method was applied to a field experiment to study eight treatments which included the applica-

tion of N, P, K and the combinations of N x P, N x K, P x K and N x P x K. It was established that increased

N contents of both wheat and barley grains stimulated phytase activities. The accumulation of P in the

grains resulted in decreases of the enzyme activities. Acid phosphatase activities in wheat and barley were

less impacted by the applied fertilizers as evidenced by small statistical differences that were established.

No specific trend of K-dependent influence on both enzymes was observed. The application of N- and P-

containing fertilizers may be used to modulate phytase activities in wheat and barley. If yielding a crop with

increased intrinsic phytase activities is needed, utilization of N-rich fertilizer is recommended.

Keywords: wheat, barley, fertilization, phytase, acid phosphatase, animal nutrition

InTRoduCTIon

Phytase and acid phosphatases in wheat and bar-

ley are the major enzymes which catalyze the release

of orthophosphate from phosphorylated substrates

(Viveros et al., 2000; Centeno et al., 2001). Inorganic

Correspondence:Vesela Chalova, [email protected]: 032-603-855 Fax: 032-644-102

phosphate is necessary for seed germination and

embryo growth (Brinch-Pedersen et al., 2002). The

levels of bioavailable phosphate are also important

when wheat and barley are used as feed ingredients

for monogastric animals (Pointillart et al., 1987). Non-

ruminants are incapable of utilizing phytate-bound

phosphorus (P) which may reach up to 80% of total P

content (Kirby and Nelson, 1988; Reddy et al., 1989;

Perney et al., 1993). Due to the importance of P to

Effect of Fertilization on Phytase and Acid Phosphatase Activities in Wheat and Barley Cultivated in Bulgaria

V. I. Chalova1, I. Manolov2, M. Nikolova3, L. Koleva1

1Department of Biochemistry and Molecular Biology, University of Food Technology, Plovdiv, Bulgaria2Department of Agrochemistry and Soil Science, Agricultural University, Plovdiv Bulgaria

3Department of Agriculture, University of Forestry, Sofia Bulgaria

Agric. Food Anal. Bacteriol. 2:103-110, 2012

104 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

animal health and the uncertainties of P bioavailabil-

ity in feed ration, farmers and animal producers are

inclined to over-supplement P containing sources to

create a margin of safety (Sutton et al., 2001). How-

ever, in countries with highly regulated animal waste

management, P overload is strictly monitored to pre-

vent pollution of soil and groundwater. In addition,

microbial phytase supplementation of animal diets,

which is meant to reduce the amount of excreted P

by making more of it bioavailable to animals, is very

often constrained by expenditure associated with

production and application of the enzyme (Afinah

et al., 2010). To more precisely formulate P levels in

animal diets and avoid elevated feed cost and envi-

ronmental pollution, while achieving optimal animal

performance, more information about the capacity

of the intrinsic orthophosphate releasing enzymes in

feed ingredients is necessary.

By studying phytate-degrading enzyme activities

in legume seeds, cereals, and cereal by-products,

Steiner et al. (2007) and Viveros et al. (2000) estimat-

ed intermediate phytase activities in cereals which,

however, varied over a wide range. Even for one

plant species, data for phytase and acid phospha-

tase activities reported by different authors varied

considerably (Viveros et al., 2000, Greiner and Egli,

2003; Steiner et al., 2007; Zarei, 2007). The variations

could be explained by differences in the varieties

used for the studies, growth conditions, and fertiliza-

tion (Liu et al., 2006; Steiner et al., 2007; 2008; Kaya

et al., 2009). Although information on phytase and

acid phosphatase activities in wheat and barley has

been published, little is known about their modula-

tion by nitrogen (N), P, and potassium (K) which are

major fertilizing agents used in agriculture to im-

prove soil quality and crop yields. The purpose of

this study was to evaluate the effect of N, P and K

fertilization on respective seed mineral contents as

related to phytase and acid phosphatase activities

in wheat and barley cultivated in Bulgaria. Although

Ca2+ and Mg2+ were not included in our fertilization

experiment, their contents in seeds were also evalu-

ated and discussed as potential determinants of the

enzyme activities (Igamnazarov et al., 1998).

MATeRIAlS And MeThodS

Experimental field design and fertiliza-tion

A field experiment was carried out in 2009/10 near

village Sadievo (42° 31’ 1.2” N, 26° 4’ 58.8” E), region

of Stara Zagora, Southern Bulgaria. Bulgarian variet-

ies wheat (Aglika) and barley (Aheloj 2) were grown in

soil type “vertisols” (pH(kcl) 5.3) which contained 16.3

mg NH4-N /kg, 14.0 mg NO3-N /kg, 2 mg P2O5/100g,

and 24 mg K2O /100g before the experiment. A ran-

domized block design method was used to study

eight treatments which included the application of

N, P, K, and combinations of N x P, N x K, P x K, and N

x P x K. The cereals grown in non-fertilized soil were

used as a control. Each treatment consisted of four

replications which were performed on plots sizing 5

x 5 m (25 m2). Ammonium nitrate, triple superphos-

phate and potassium chloride were applied to field

to supply N, P2O5 and K2O at the rates of 100 kg/ha,

120 kg/ha, and 80 kg/ha for the wheat, and 80 kg/ha,

120 kg/ha, 80 kg/ha for the barley respectively.

Cereals (wheat and barley) were field collected

and air dried. Representative samples (100 g) were

taken, ground to pass a 1 mm sieve and stored in

sealed containers. All reagents used throughout the

experiments were of analytical grade and bought

from Sigma (Buchs, Switzerland).

Determination of N, P, K, Ca, and Mg in cereal grains

Seed samples were digested with concentrated

HNO3 and heated in a MarsXpress microwave diges-

tion system (CEM GmbH, Germany) to determine P,

K, Ca, and Mg contents. Concentration levels of K,

P, Ca, and Mg were established by using an Opti-

cal Emission Spectrometry with Inductively Coupled

Plasma (ICP OES), Liberty Series II at 769.896 nm,

213.618 nm, 315.887 nm, and 279.079 nm respective-

ly. N was determined by Kjeldahl method (Bremner,

1996).

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 105

Enzyme activity measurements

Intrinsic phytase activities in wheat and barley

were determined by the extraction procedure of

Grainer and Egli (2003) with some modifications. The

method is based on the quantification of inorganic

phosphate released by phytase from phytate which

was used as a substrate. The liberated phosphate

was measured according to the ammonium-molyb-

date method (James, 1999). Briefly, samples (3 g)

were extracted for 30 min with 100 ml 0.25 M acetate

buffer solution (pH 5) at constant stirring and room

temperature (22ºC). Solid particles were removed

by centrifugation for 15 min at 6000 g (MPW Med.

Instruments, Warsaw, Polska) and supernatants were

analyzed for phytase and acid phosphatase enzyme

activities. For the phytase evaluation, the reaction

mixture consisted of 0.9 ml acetate buffer (0.25 M,

pH 5), 2 ml 7.5 mM sodium phytate (Sigma-Aldrich

P8810), and 0.1 ml test solution was incubated for 30

min at 37ºC. The enzyme reaction was stopped by

adding a stop solution (2:1:1 v/v/v) consisted of nitric

acid (1:2 v/v nitric acid:water), ammonium molybdate

(5%), and ammonium metavanadate (0.235%). The

yellow complex, formed after the reaction between

the liberated inorganic phosphate and the acidic

molybdate/vanadate reagent, was measured with

a spectrophotometer (Carl Zeiss, Jena, Germany)

at 415 nm. Phytase activity was calculated against a

standard curve constructed with potassium dihydro-

gen phosphate and expressed as unit/kg (U/kg) on a

dry matter (DM) basis. One phytase unit was defined

as the amount of the enzyme which liberates 1 µmol

of inorganic phosphorus per minute from 5 mmol of

sodium phytate at pH 5 and 37ºC.

Acid phosphatase activity was determined as de-

scribed by Zyla et al. (1989). The method is based

on the quantification of the p-nitrophenol released

from p-nitrophenyl phosphate by the catalytic action

of the enzyme. Reaction mixture contained 1 ml 10

mM substrate (p-nitrophenyl phosphate) dissolved

in 0.25 M acetate buffer (pH 5) and 0.2 ml extracted

enzyme. After incubation for 30 min at 37 ºC, the re-

action was stopped by the addition of 5 ml 50 mM

NaOH. The intensity of yellow color was measured

with a spectrophotometer and the enzyme activity

was calculated against a standard curve constructed

with graded concentrations of p-nitrophenol. One

unit acid phosphatase activity was defined as the

amount of the enzyme which liberates 1 µmol of p-

nitrophenol per minute under above conditions.

Variability, replication, and statistical analysis

Statistical analysis was performed using the Sta-

tistical Package for the Social Sciences (SPSS) pro-

gram (IBM SPSS Stattistics 17, Somers, NY, USA).

Presented results are averaged means of at least two

independent experiments ± standard deviations.

Mean differences and between-subject effect were

established by one-way analysis of variance (ANO-

VA) using the general linear model procedure and

Duncan’s multiple comparison test. Statistical differ-

ences were considered significant at p < 0.05.

ReSulTS And dISCuSSIon

Effect of fertilization on mineral con-tents of wheat and barley grains

Plant fertilization is one of the most important in-

tensifying factors of wheat and barley crop produc-

tion. In addition to crop yield, it influences the qual-

ity of harvested product which may include mineral

and protein contents, chlorophyll and carotenoides,

amino acid composition and enzyme activities (Ko-

szanski et al., 1997; Manolov et al., 1999; Černý et

al., 2010). N, P and K are macronutrients which are

required by plants in relatively large amounts. They

are commonly included in agricultural practices as

fertilizing agents to improve soil fertility and modu-

late plant enzyme activities towards desired crop

characteristics (Stewart et al., 2004; Kaya et al., 2009;

Balabanli et al., 2010).

In our study, the application of N-containing fer-

tilizers in all cases (N, NP, NK and NPK) resulted in

increases of N contents of both wheat and barley

grains compared to controls (Tables 1 and 2). Av-

106 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

erage increments of 19.38% and 19.53% in grain N

concentrations in response to N fertilization were es-

tablished for wheat and barley respectively. Our data

agreed with Bettaieb-Ben Kaab et al. (2006) who re-

ported increased concentrations of N in barley grains

compared to control in response to all levels of N

fertilization which included 40, 80, and 120 kg/ha.

Albrizio et al. (2010) reported up to 15.8% average

increases in N contents of wheat and barley grains as

consequence of N fertilization which is close to our

findings. Our results were not unexpected because

of plant capabilities to accumulate N upon exces-

sive N soil availability (Golik et al., 2005). According

to Przulj and Momčilović (2003), N is accumulated in

Table 1. Effect of fertilization differences on mineral content of wheat grains

Fertilization, kg/ha

N, % P, % K, % Ca, % Mg, %

control 1.78 ± 0.15bc 0.24 ± 0.08b 0.41 ± 0.03 0.046 ± 0.008b 0.105 ± 0.009

N100 2.14 ± 0.12a 0.21 ± 0.02c 0.39 ± 0.03 0.048 ± 0.004b 0.093 ± 0.006

P120 1.65 ± 0.12c 0.29 ± 0.03a 0.38 ± 0.05 0.043 ± 0.001b 0.97 ± 0.004

K80 1.75 ± 0.17 bc 0.24 ± 0.07b 0.35 ± 0.02 0.042 ± 0.009b 0.107 ± 0.002

N100P120 2.16 ± 0.20a 0.25 ± 0.02b 0.39 ± 0.04 0.039 ± 0.004b 0.110 ± 0.002

N100K80 2.04 ± 0.09ab 0.24 ± 0.09b 0.38 ± 0.02 0.045 ± 0.006b 0.105 ± 0.001

P120K80 1.63 ± 0.10c 0.29 ± 0.03a 0.42 ± 0.09 0.083 ± 0.006a 0.104 ± 0.004

N100P120K80 2.16 ± 0.11a 0.32 ± 0.04a 0.38 ± 0.04 0.043 ± 0.002b 0.101 ± 0.003

Data represent average means of at least two independent experiments ± standard deviations. a-c Means in a column with different superscripts differ significantly (p<0.05). Data for K and Mg were not found

significantly different (p>0.05).

Table 2. Effect of fertilization differences on mineral content of barley grains

Fertilization, kg/ha N, % P, % K, % Ca, % Mg, %

control 1.51 ± 0.13b 0.23 ± 0.10cd 0.32 ± 0.03d 0.049 ± 0.004c 0.107 ± 0.009

N80 1.78 ± 0.15a 0.22 ± 0.03cd 0.39 ± 0.02ab 0.047 ± 0.003c 0.115 ± 0.001

P120 1.44 ± 0.18bc 0.28 ± 0.04ab 0.40 ± 0.02a 0.055 ± 0.002c 0.124 ± 0.001

K80 1.39 ± 0.11c 0.24 ± 0.02c 0.37 ± 0.03bc 0.051 ± 0.002c 0.107 ± 0.003

N80P120 1.90 ± 0.26a 0.24 ± 0.09c 0.34 ± 0.04bc 0.039 ± 0.004d 0.109 ± 0.002

N80K80 1.91 ± 0.11a 0.20 ± 0.02d 0.35 ± 0.03c 0.045 ± 0.006cd 0.100 ± 0.001

P120K80 1.31 ± 0.07c 0.30 ± 0.09a 0.43 ± 0.02a 0.072 ± 0.001b 0.116 ± 0.002

N80P120K80 1.63 ± 0.30a 0.26 ± 0.07b 0.39 ± 0.02ab 0.131 ± 0.002ab 0.118 ± 0.006

Data represent average means of at least two independent experiments ± standard deviations. a-d Means in a column with different superscripts differ significantly (p<0.05). Data for Mg were not found sig-

nificantly different (p>0.05).

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 107

wheat and barley during vegetation period to pro-

vide optimal protein and nucleic acid syntheses and

is translocated to kernel during reproductive phase.

Positive response of N content of wheat and barley

grains to N fertilization was also reported by Koszan-

ski et al. (1997), and Małecka and Blecharczyk (2008).

Phosphate-based fertilization exhibited similar

trend of increase in P concentrations in both wheat

and barley (Tables 1 and 2). Except for the combined

fertilization with P and N, the grains, collected from

plots enriched with P, PK and NPK, contained sig-

nificantly higher P concentrations than non-fertilized

grains (p< 0.05). Our data agreed with Syltie and

Dahnke (1983) who also observed increased P-con-

tent responses of two hard red spring wheat culti-

vars to incremented application of a P-rich fertilizer

to soil.

Fertilization of wheat with K80 did not influenced

K contents of grains. No significant differences in K

grain concentrations (p< 0.05) among all treatments

were observed (Table 1). This is probably due to

relatively high initial content of K in soil (24 mg K2O

/100g) before starting the experiment. When wheat

is grown in soil containing optimal K concentrations,

further increase of K availability may not affect K

plant content (Slaton et al., 2008). In barley, signifi-

cant differences among K contents of grains were

established but they were ambiguously related to K

dose applied to soil. They were rather due to inter-

relationships of macronutrients where P (P, NP, PK,

and NPK) exerted the most stimulating effect on K

accumulation in barley grains (Table 2).

Although Ca and Mg ions were not included in

our field experiment as fertilizers, they were quanti-

tatively measured because of possible inter-element

relationships (Markert, 1993; Li et al., 2010) and po-

tential influence on phytase and acid phosphatase

activities. Indeed, Ca contents of both wheat and

barley subjected to the experimental fertilization de-

sign were found significantly different with PK and

NPK contributing the most. None of the fertilizers or

their combinations influenced Mg contents of wheat

and barley grains.

Effect of fertilization on phytase and acid phosphatase activities in wheat and barley grains

In our study, nitrogen fertilization (N and NK) and

the respective high N contents in seeds (Table 1 and

2) stimulated phytase activities in both wheat and

barley (Table 3 and 4). Similarly, Kaya et al. (2009) es-

tablished positive effect of N fertilization on phytase

activity in chickpea (Akcin 91) as evidenced by the in-

creased activity of the enzyme (4.3 U/g) compared to

control (3.8 U/g) in a response to soil fertilization with

N at the level of 60 kg N/ha. According to Eastwood

and Laidman (1971), the increased phytase levels at

higher N availability is mediated by certain nitrogen-

containing compounds including glutamine, purine

and pyrimidine nucleotides. Although significantly

not different from the controls, highest acid phos-

phatase activities (absolute values) were estimated

in wheat (6 529 ± 130 U/kg) and barley (4189 ± 254

U/kg) seeds containing highest N concentrations.

Increasing acid phosphatase activities from 5.27 to

7.87 mmol/kg in wheat leaves which corresponded

to increasing N levels ranging from 13.1 to 45.4 mg

N-NO3/kg were observed by Koszanski et al. (1997).

In contrast to N, the wheat and barley seeds con-

taining highest P concentrations (P, PK, NPK; Table 1

and 2) exhibited the lowest levels of phytase activi-

ties (Table 3 and 4). Although the biological function

of phytase is to liberate inorganic P to provide suf-

ficient amounts for germinating seeds and growing

plants, it looks that excessive P availability may inhib-

it the enzyme. By studying the control mechanisms

of the phytin-phytase system in wheat embryos,

Sartirana and Bianchetti (1967) established that the

rate of phytin breakdown was controlled in vivo by

the concentration of inorganic phosphate, through

the inhibition of phytase activity. Statistical decrease

of acid phosphatase activity caused by enhanced P

concentrations was established only for barley fertil-

ized with P120K80 (Table 4).

In addition to P concentrations, Ca ions may also

contribute to phytase inhibition. In fact, wheat seeds

fertilized with P120K80 resulted in highest accumula-

tion of Ca (0.083 ± 0.006%) and one of the lowest lev-

108 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

els phytase- and acid phosphatae activities (Table 3).

Similarly, the barley seeds containing 0.072 ± 0.001%

Ca (Table 2) exhibited phytase- and acid phosphatae

activities significantly lower than the control (Table 4).

According to Igamnazarov et al. (1998), the influence

of Ca ions on phytase activity is dose-dependent.

Low Ca concentrations (1x10-5 M) did not influence

phytase activity in cotton plants. However, higher Ca

concentrations (5x10-5 M) reduced phytase activity

by 12%.

ConCluSIonS

Overall mineral soil fertilization influenced phy-

tase and acid phosphatase activities in both wheat

and barley grains. Acid phosphatase activities were

less impacted as evidenced by small statistical differ-

ences that were established. While the increased N

contents of seeds stimulated phytase activities, the

abundance of P negatively controlled the liberation

of phytine-bound P caused by phytase. No specific

trend of K influence on both enzymes was estab-

lished. Therefore, the application of N- and P-con-

taining fertilizers may be used to modulate phytase

activities in wheat (Aglika) and barley (Aheloj 2). If

yielding a crop with increased intrinsic phytase ac-

tivities is needed, utilization of N-rich fertilizer may

be the choice.

ACknowledgeMenTS

This research was supported by project “Best

Management Practices for Sustainable Crop Pro-

duction in Bulgaria” financed by International Plant

Nutrition Institute (IPNI) USA and K+S KALI GmbH

Germany.

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Fertilization, kg/ha

Phytase Activity, U/kg

Acid Phosphatase Activity, U/kg

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N100 2349 ± 75b 6529 ± 130 a

P120 1160 ± 93f 5243 ± 292 b

K80 1691 ± 137cd 5283 ± 322 ab

N100P120 2168 ± 90b 5317 ± 253 ab

N100K80 3455 ± 184a 5331 ± 224 ab

P120K80 1593 ± 112cd 5246 ± 139 b

N100P120K80 1478 ± 34de 6565 ± 311 a

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Acid Phosphatase Activity, U/kg

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P120 1410 ± 85d 3664 ± 40abc

K80 3442 ± 316a 3778 ± 106abc

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N80K80 3372 ± 227ab 3583 ± 170bc

P120K80 1564 ± 42d 3509 ± 35c

N80P120K80 2763 ± 235bc 3849 ± 205abc

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www.afabjournal.comCopyright © 2012

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Mixed populations of domesticated and feral pig gut microorganisms (Recombined Porcine-Derived

Continuous Flow culture; RPCF and Feral Culture; FC, respectively) were grown in continuous culture to

investigate the effects of tylosin on antimicrobial resistance. Cultures established in steady state were in-

oculated with 9.7 log10 colony forming units (CFU) of a tylosin-resistant Enterococcus faecium and allowed

7 days to re-establish equilibrium before administration of 100 µg tylosin mL-1. Total culturable anaerobes

recovered on non-antibiotic supplemented medium, thus inclusive of tylosin-sensitive and -insensitive bac-

teria, ranged from 7.15 to 9.20 log10 CFU mL-1 throughout 8 days of tylosin administration and 6 subse-

quent days without tylosin administration. Recovery of total anaerobes on tylosin-supplemented medium

revealed that populations of total tylosin-insensitive anaerobes ranged from 6.30 to 9.02 log10 CFU mL-1

during the experiment. Concentrations of the introduced tylosin-resistant E. faecium decreased to near

minimum detectable levels (1.3 log10 CFU mL-1) in the cultures before initiation of tylosin administration

and then increased to 6.80 ± 0.28 and 8.30 ± 0.43 log10 CFU mL-1 in RPCF and FC cultures, respectively,

and remained higher than day 0 concentrations for the remainder of the experiment. Endogenous tylosin-

insensitive Enterococcus were undetectable before administration of tylosin but tylosin-resistant E. faecalis

and E. hirea found to have acquired an ermB gene of expected size and sequence of that contained in the

introduced E. faecium were enriched to 7.74 ± 0.37 and 3.85 ± 4.03 after initiation of tylosin administra-

tion. These results demonstrate the acquisition, propagation and persistence of tylosin-resistance in mixed

populations of domestic and feral swine gut microflora.

Keywords: Anaerobic bacteria, antimicrobial resistance, antimicrobial resistance transfer, continu-

ous flow culture, domestic swine, Enterococcus, feral swine, gut microflora, macrolide, tylosin. bac-

terial activity

Correspondence: Robin C. Anderson, [email protected]: +1 -979-260-9317 Fax: +1-979-260-9332

Transfer of tylosin resistance between Enterococcus spp. during continuous-flow culture of feral or domestic porcine gut microbes†

N. Ramlachan1,2, R.C. Anderson1, K. Andrews1, R.B. Harvey1 and D.J. Nisbet1

1United States Department of Agriculture/Agricultural Research Service, Food & Feed Safety Research Unit, College Station, Texas, USA

2Current address: Department of Biosciences, Agriculture and Food Technology, University of Trinidad and Tobago, Piarco, Trinidad, WI

† Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warrenty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.

Agric. Food Anal. Bacteriol. 2:111-120, 2012

112 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

InTRoduCTIon

Macrolide antibiotics are commonly used in hu-

man and veterinary medicine, primarily to treat in-

fections caused by Gram-positive bacteria (Gaynor

and Mankin, 2003). In the swine industry, the mac-

rolide antibiotic tylosin is also used as a feed addi-

tive to improve production efficiency (Gaynor and

Mankin, 2003). Concern exists, however, that the

continued agricultural use of antibiotics, especially

their sub-therapeutic use for growth promotion, is

contributing to the emergence and proliferation of

microbial populations resistant to many antibiotics

used to treat medically important diseases (Aar-

estrup, 2005). For macrolide antibiotics, resistance

can occur via acquisition of erm methyltransferases,

which catalytically inactivate the macrolide’s target-

ed binding site, via acquisition of multidrug efflux

pumps or even, albeit infrequently, via point muta-

tions in the microorganism’s genome or inactivation

of the macrolide (Chopra and Roberts, 2001; Gaynor

and Mankin, 2003; Karlsson et al., 2004; Poole, 2005).

Recovery of bacteria harbouring erm genes from

domestic conventionally-raised and non-antibiotic-

raised swine as well as from swine production habi-

tats has been reported (Chee-Sanford et al., 2001;

Jackson et al., 2004; Wang et al., 2005). Likewise,

macrolide resistance in gut bacteria from undomes-

ticated swine has also been reported (Ramlachan et

al., 2007; Stanton and Stoffregen, 2004). However, it

is still not clear just how easily that native popula-

tions of gut bacteria may acquire macrolide resis-

tance and how long acquired resistance may per-

sist within affected populations once the selective

pressure of the antibiotic is removed. For instance,

an immediate increase in proportions of macrolide-

resistant enterococci was observed in pigs following

initiation of subtheapeutic feeding of tylosin to pigs

(Aarestrup and Carstensen, 1998).

More recently, continuous flow cultures estab-

lished with mixed populations of gut bacteria origi-

nating from domesticated and feral swine were used

to assess the effects of low (25 µg mL-1) and high (100

µg mL-1) amounts of tylosin administration on select

populations of resident bacteria (Ramlachan et al.,

2008). Results from that study indicated numbers of

an exogenously introduced macrolide-resistant En-

terococcus faecium were rapidly enriched in continu-

ous flow cultures during treatment with 100 µg tylo-

sin mL-1 but high numbers of the resistant bacterium

were maintained only during tylosin administration.

Moreover, results revealed that tylosin administra-

tion differentially selected for tylosin resistant bacte-

ria from within the autochthonous populations, with

acquisition of tylosin resistance appearing within en-

dogenous Enterococcus spp. in the population from

the domestic pig but not in the population from the

feral pig. However, the source of this resistance ac-

quisition by the endogenous Enterococcus spp. in

the domestic pig culture was not determined. Con-

sequently, the present experiment was conducted

to further investigate the potential development,

propagation, persistence and transfer of resistance

elements within the enterococcal community in the

respective continuous flow cultures.

MATeRIAlS And MeThodS

Continuous flow culture establishment

The two separate mixed populations of porcine

gut bacteria had been used and characterized in

earlier studies and were established in continuous

flow culture as previously described (Harvey et al.,

2002; Hume et al., 2001; Ramlachan et al., 2008). The

culture defined as Recombined Porcine-Derived

Continuous Flow (RPCF) culture had been previ-

ously established with cecal contents obtained from

a traditionally reared domestic pig and the culture

defined as our Feral culture (FC) was established un-

der similar conditions with cecal contents from an

adolescent feral boar. Both cultures were established

and maintained as parent cultures in BioFlo chemo-

stats (New Brunswick Scientific Company, Edison,

NJ) with culture volumes of 550 ml. The culture me-

dium was Viande Levure broth (Barnes et al., 1979)

which was prepared and maintained anaerobically

under a stream of carbon dioxide and infused at 0.40

ml/min which corresponds to a 24 h vessel turnover.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 113

media were incubated 48 to 72 h at 37°C in a Bac-

tron Anaerobic Chamber (Sheldon Labs Manufactur-

ing Inc., Cornelius, OR) and colonies enumerated

on agar medium without tylosin selection include

those in the population that are both sensitive and

insensitive to tylosin; colonies enumerated on agar

medium with 100 µg tylosin mL-1 are only those in

the population that are tylosin-insensitive. For con-

firmation of tylosin resistance, 10 tylosin-insensitive

colonies per population were subsequently selected

and tested as previously described (Ramlachan et al.,

2008). Specific identification of bacteria from select

colonies was achieved using rapid ID 32 STREP, rap-

id 20E, 20NE, 20A, and rapid ID 32 A identification

strips (bioMérieux, Hazelwood, MO). Indole spot

tests (Anaerobe Systems, Morgan Hill, CA), E-testTM

(AB Biodisk, Piscataway, NJ) and fermentation acid

production determined via gas chromatography

(Hinton et al., 1990) were also used in this analysis.

PCR to confirm transfer of resistance genes

Bacteria were lysed with equal volume of 0.2%

(w/v) of Triton X-100 and heated to boiling at 100°C

for 5 min in water bath, allowed to cool and subse-

quently used as templates for PCR. Specific primers

for the following genes: ermB, ermC, ermF, ermG

and ermQ (see Table 1) were used for amplifications

as previously published (Bendle et al., 2004; Frye et

al., 2006; Löfmark et al., 2006; Perrin-Guyomard et

al., 2005). Amplification was carried out on a MJ ther-

mocycler Model PTC200 (Fisher Scientific, Hampton,

NH) using the following conditions: 5 min at 94°C; 35

cycles of 94°C for 1 min, 55 °C for 1 min and 72 °C for

2 min. All PCR products were analyzed by gel elec-

trophoresis (1% agarose in 1x TAE buffer), stained

with ethidium bromide and visualized by UV light.

Sizes of products were determined by comparing

them with a 100 bp ladder (New England Biolabs,

Ipswich, MA).

Cultures were incubated at 39°C and agitated at 100

rpm. Once established in steady state, these respec-

tive parent cultures contained representative species

of genera common to the pig gut (Robinson et al.,

1981) including but not limited to Bacteroides, Clos-

tridium, Enterococcus and Streptococcus (Harvey et

al., 2002; Ramlachan et al., 2008). Of these, B. unifor-

mis (possessing ermG) from culture RPCF and C. ha-

thewayi (possessing ermF) from culture FC showed

resistance to tylosin at MIC >512 µg mL-1; with B. uni-

formis being prominent within culture RPCF and C.

hathewayi being the predominant anaerobe recov-

ered from culture FC (Ramlachan et al., 2007; 2008).

Neither culture contained endogenous Enterococ-

cus spp. with measurable tylosin resistance.

Both parent cultures were used to provide inocula

(10% vol/vol) to establish separate RPCF and FC test

cultures, in duplicate, which after 7 days were each

inoculated with 5 x 109 colony forming units (CFU) of

a multi-drug resistance E. faecium strain so as to pro-

vide an exogenous gene donor exhibiting resistance

to tylosin. The E. faecium strain used here, which was

phenotypically distinguishable from Enterococcus

spp. endogenous to the mixed populations in the

test cultures, as described previously (Ramlachan et

al., 2008). The respective cultures were each allowed

another 7 days culture to re-establish equilibrium as

evidenced by the gradual decline of the inoculated

E. faecium strain, which was present below our limit

of detection (10 CFU mL-1 by day 0). The cultures

were then continually infused with culture medium

containing 100 µg tylosin mL-1 for 8 days, which was

immediately followed by 6 days of infusion of me-

dium lacking tylosin. Collection of fluid samples from

the test cultures began on day -3 relative to tylosin

treatment and continued during 8 days of tylosin

administration and 6 days of tylosin withdrawal. Col-

lected samples were quantitatively cultured, via plat-

ing of 10-fold serial dilutions, to recovery media that

had been prepared with or without 100 µg tylosin

mL-1. Recovery media were anaerobic Brucella blood

agar (Anaerobe Systems, Morgan Hill, CA), for de-

tection of total anaerobes and M Enterococcus (ME)

agar (Becton Dickinson and Company, Sparks, MD)

for detection of Enterococcus spp. Inoculated agar

114 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

Statistical analysis

Log10 transformations of bacterial concentrations

obtained from duplicate cultures were analyzed for

main effects of culture type (RPCF versus FC culture)

and day of cultivation on days 0 through the end of

the experiment using a repeated measures analysis

of variance (Statistix 9 Analytical Software, Tallahas-

see, FL). Samples yielding no detectable colonies of

bacteria were assigned a limit of detection value of

1.3 log10 CFU mL-1 which was our limit of detection.

Comparison of means to values observed on day 0

for each of the respective cultures was accomplished

using a Two Sided Dunnett’s Multiple Comparison

procedure with P < 0.05.

ReSulTS And dISCuSSIon

Continuous flow culture of intestinal microorgan-

isms has been used to study competitive interac-

tions between commensal and pathogenic micro-

flora (Harvey et al., 2002; Hume et al., 2001; Nisbet

et al., 2000) as well as to investigate potential fac-

tors affecting spontaneous acquisition of antibiotic

resistance (Kim et al., 2005). In the present experi-

ment, populations of total culturable anaerobes re-

covered on non-antibiotic supplemented Brucella

blood agar, thus representing numbers of both ty-

losin-sensitive and –insensitive populations, did not

differ (P = 0.30; SEM = 0.13) between the RPCF or

FC cultures, ranging from 7.15 to 9.20 log10 CFU mL-1

throughout the experiment (Figure 1). When tested

for main effects of day, concentrations of total cultur-

able anaerobes were affected only marginally within

the RPCF cultures (P = 0.041) and were not affected

in the FC cultures (P = 0.058) (Figures 1). Moreover,

effects of tylosin on total culturable anaerobes in the

mixed populations were not readily apparent as vi-

able cell counts observed after initiation of tylosin

administration did not differ from those populations

measured on day 0. Populations of total culturable

tylosin-insensitive anaerobes were recovered on

tylosin-supplemented Brucella blood agar and were

observed in both RPCF and FC cultures even before

administration of tylosin administration (Figure 1),

likely due to the presence of the ermG-containing B.

uniformis and the ermF-containing C. hathewayi en-

Table 1. Oligonucleotide primers used in PCR amplification of Enterococcus spp. isolate.

Gene Primer Sequence (5’-3’) Accession Number

Reference

ermB F: TAACGACGAACCTGGCTAAAAT

R: ATCTGTGGTATGGCGGGTAAGAJ243541 Frye et al., 2006

ermC F: AGTACAGAGGTGTAATTTCG

R: AATTCCTGCATGTTTTAAGGNC001386 Frye et al., 2006

ermF F: GCCAACAATGTTGTTGTT

R: CGAAATTGTCCTGACCTGN/A Bendle et al., 2004

ermG F: ACTGCTGAATTGGTAAAGAGATG

R:TGTGCTTATGTTGTAAGGTATGCN/A Löfmark et al., 2006

ermQ F: CACCAACTGATATGTGGC

R: CAATCTACACTAGGCATGN/A Bendle et al., 2004

vatA F: ATAATGAATGGAGCAAACCATAGGATG

R: ACCAATCCAAACATCATTACCN/A

Perrin-Guyomard et al., 2005

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 115

Figure 1. Recovery of total culturable anaerobes (A; containing both tylosin-sensitive and -insensi-tive bacteria) and of tylosin-insentive anaerobes (B) during continuous flow culture of mixed popu-lations (in duplicate) of porcine gut bacteria obtained from a domestic (culture RPCF, circles) or a feral (culture FC, squares) swine. Cultures were experimentally innoculated on day -7 with an exogenous tylosin-resistant Enterococcus faecium. Tylosin administration (100 µg mL-1) began af-ter sampling on day 0 and ceased after sampling on day 8 (as indicated by arrow). Bacteria were quantitatively recovered on Brucella blood agar supplemented without (A) or with 100 µg tylosin mL-1 (B). Tests for differences in bacterial concentrations on days 0 through 14 were accomplished using a repeated measures analysis of variance; asterisks indicate daily concentrations that differ from concentrations observed on day 0 for the respective cultures (SEM = 0.29 and 0.30 for total anaerobes in RPCF and FC cultures, respectively; SEM = 0.43 and 0.35 for tylosin-insensitive an-aerobes in RPCF and FC cultures, respectively).

0

2

4

6

8

10

12

-3 0 3 6 9 12 15

Log 1

0C

FU m

l-1

Day of incubation

Total anaerobes RPCF cultureTotal anaerobes FC culture

A

0

2

4

6

8

10

12

-3 0 3 6 9 12 15

Log 1

0C

FU m

l-1

Day of incubation

Tylosin-insensitive anaerobes RPCF cultureTylosin-insensitive anaerobes FC culture

* *

B

116 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

dogenous to the RPCF and FC cultures, respectively.

Concentrations of total culturable tylosin-insensitive

anaerobes ranged from 6.30 to 9.02 log10 CFU mL-1

and exhibited similar concentration curves as those

observed for total culturable anaerobes (Figure 1).

Consequently, a main effect due to the different cul-

tures was not observed (P = 0.20; SEM = 0.17). Main

effects of day of cultivation were observed on popu-

lations of total culturable tylosin-insensitive anaer-

obes recovered from the RPCF (P = 0.049) and the

FC cultures (P = 0.027) but when compared to num-

bers measured on day 0, the total culturable tylosin-

insensitive anaerobes differed only on days 3 and 4

of tylosin treatment in the RPCF culture (Figure 1).

Populations of the exogenous tylosin-resistant

E. faecium, which characteristically developed into

small, light pink colonies, were inoculated to achieve

6.95 log10 CFU mL-1 in the RPCF and FC cultures 7

days before initiation of tylosin administration. Num-

bers of the exogenous tylosin-resistant E. faecium

declined rapidly soon after inoculation to near or be-

low our level of detection (1.3 log10 CFU mL-1) by day

0 (Figure 2). A main effect of culture on numbers of

exogenous tylosin-resistant E. faecium recovered on

days 0 through 14 was not observed (P = 0.065; SEM

= 0.26) although a main effect of day was observed

for both cultures (P = 0.0001 and < 0.0001 for RPCF

and FC cultures, respectively). In both cases, num-

bers of tylosin-resistant E. faecium began increas-

ing soon after initiation of tylosin administration

Figure 2. Recovery of an experimentally introduced (on day -7) exogenous tylosin-resistant En-terococcus faecium during continuous flow culture of mixed populations (in duplicate) of porcine gut bacteria obtained from a domestic (culture RPCF, circles) or a feral (culture FC, squares) swine. Tylosin administration (100 µg mL-1) began after sampling on day 0 and ceased after sampling on day 8 (as indicated by arrow). Bacteria were quantitatively recovered on M Enterococcus agar supplemented with 100 µg tylosin mL-1. Tests for differences in bacterial concentrations on days 0 through 14 were accomplished using a repeated measures analysis of variance; asterisks indicate daily concentrations that differ from concentrations observed on day 0 for the respective cultures (SEM = 0.60 and 0.58 for RPCF and FC cultures, respectively).

0

2

4

6

8

10

12

-3 0 3 6 9 12 15

Log 1

0C

FU m

l-1

Day of incubation

Tylosin-resistant Enterococcus faecium RPCF culture Tylosin-resistant Enterococcus faecium FC culture

*

*

* * * ** * *

***

* * ** * * *

*

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 117

Figure 3. Recovery of an endogenous tylosin-resistant Enterococcus during continuous flow cul-ture of mixed populations (in duplicate) of porcine gut bacteria obtained from a domestic (culture RPCF, circles) or a feral (culture FC, squares) swine. Cultures were experimentally innoculated on day -7 with an exogenous tylosin-resistant Enterococcus faecium. Tylosin administration (100 µg mL-1) began after sampling on day 0 and ceased after sampling on day 8 (as indicated by arrow). Bacteria were quantitatively recovered on M Enterococcus agar supplemented with 100 µg tylosin mL-1. Tests for differences in bacterial concentrations on days 0 through 14 were accomplished using a repeated measures analysis of variance; asterisks indicate daily concentrations that differ from concentrations observed on day 0 for the respective cultures (SEM = 0.73 and 0.95 for RPCF and FC cultures, respectively).

0

2

4

6

8

10

12

-3 0 3 6 9 12 15

Log 1

0C

FU m

l-1

Day of incubation

Tylosin-resistant endogenous Enterococcus spp. RPCF culture Tylosin-resistant endogenous Enterococcus spp. FC culture

*

* *

* * **

* * **

and although numbers began to decline gradually

after day 6 of treatment they remained higher than

numbers recovered on day 0 for the duration of this

experiment (Figure 2). These findings are consistent

with those observed in our earlier study (Ramlachan

et al., 2008) which, except for a 2 log10 lower inocu-

lation of the tylosin-resistant E. faecium, was con-

ducted similarly to this study. Thus, it appears that

the tylosin resistance in this E. faecium may have

been inducible only in the presence of the antibiotic

or that in the absence of the selective pressure of

tylosin; this tylosin-resistant E. faecium may be less

competitive in the mixed populations.

At the beginning of the present experiment, pop-

ulations of endogenous tylosin sensitive Enterococ-

cus spp., which developed into large and deep red

colonies, achieved concentrations of 7.63 ± 1.0 and

5.65 ± 1.85 log10 CFU mL-1 in the RPCF and FC cul-

tures. Endogenous tylosin-insensitive Enterococcus

spp., were not detected from among these popu-

lations prior to administration of tylosin. Beginning

as soon as 2 days after the start of tylosin adminis-

tration, however, numbers of tylosin-insensitive En-

terococcus colonies exhibiting morphological char-

acteristics indistinguishable from the endogenous

Enterococcus spp. yet clearly distinguishable from

118 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

the exogenous tylosin-resistant E. faecium began

increasing in both of the duplicate RPCF cultures

and in one of the FC cultures. Although numbers of

tylosin-insensitive Enterococcus recovered on days

0 through 14 did not differ between the RPCF and

FC cultures (P = 0.067, SEM = 0.65), a significant ef-

fect of day of cultivation was observed for the RPCF

cultures (P < 0.0001) but not for the FC cultures (P =

0.500) (Figure 3). These populations, which we sus-

pected were derived from the endogenous popula-

tion, achieved a maximum concentration in the RPCF

cultures after 7 days of treatment and persisted at >

6.00 log10 CFU mL-1 even during withdrawal (Figure

3). Populations of tylosin-insensitive Enterococcus

spp. similarly indistinguishable from the endoge-

nous Enterococcus spp. emerged in the FC culture

but not until after 7 days of tylosin administration

(Figure 3). The population in the FC culture persisted

during the tylosin withdrawal period but at a much

lower density than those in the RPCF cultures (Figure

3). Further tests of representative isolates confirmed

that these Enterococcus spp. identified as E. faeca-

lis from the RPCF cultures and as E. hirea from the

FC culture, were indeed tylosin-resistant. Moreover,

PCR amplification of DNA extracted from the en-

dogenous tylosin-resistant Enterococcus spp. from

both RPCF and FC cultures produced PCR products

of the expected size and sequence of the ermB gene

amplicon obtained for the exogenous E. faecium

(Figure 4). However, PCR products for ermC, ermF,

ermG and ermQ were not detected from these en-

dogenous tylosin-resistant Enterococcus spp. These

results suggest that the endogenous enterococci

had acquired resistance to tylosin via acquisition of

the ermB gene from E. faecium. Whereas it is attrac-

tive to speculate that this acquisition occurred most

simply via direct genetic exchange between these

Gram-positive bacteria, we cannot exclude the pos-

sibility of other bacterial hosts may have served as

intermediaries in this transfer.

Figure 4. A PCR product was obtained for ermB (311bp) at the expected size for select colonies of Enterococcus faecium (lane 2), Enterococcus hirea from FC (lane 4) and Enterococcus faecalis from RPCF (lane 5). All colonies were grown on M Enterococcus agar supplemented with 100 µg tylosin mL-1. Colonies tested at the start of the experiment showed no evidence that Enterococcus spp. from the FC (lane 1) or the RPCF (lane 7) possessed the ermB gene. Lanes 3 and 6 are the marker lanes illustrating the product obtained to be approximately 311 bp.

1 2 3 4 5 6 7

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 119

In conclusion, results from this study showed that

tylosin administration differentially promoted en-

richment of tylosin-insensitive bacterial populations

within continuous flow cultures of mixed popula-

tions of gut bacteria derived from gut contents of

a traditionally reared domesticated pig (RPCF) and

a feral pig (FC). In the initial absence of the selec-

tive pressure of 100 µg tylosin mL-1, populations of

an introduced exogenous tylosin-resistant E. fae-

cium declined and did not reappear at substantial

numbers until after the initiation of tylosin adminis-

tration. Conversely, populations of endogenous tylo-

sin-resistant Enterococcus spp., while initially absent

or at undetectable concentrations in the mixed cul-

ture populations, were enriched in cultures derived

from both the domestic and feral pig. However,

once enriched the endogenous tylosin-resistant En-

terococcus populations persisted even during with-

drawal of the selective pressure of tylosin. Results

from our study also provided evidence that at 100

µg tylosin mL-1, genetic transfer of the tylosin resis-

tance occurred between the exogenous E. faecium

introduced to the cultures and the endogenous En-

terococcus spp., as the resistance gene, known as

ermB, was found in the newly-resistant endogenous

Enterococcus spp. These findings have implications

for transfer of antibiotic resistance in other species,

showing that even in feral populations which can be

assumed to have low levels of previous antibiotic ex-

posure; resistance can be obtained in endogenous

bacterial populations after exposure to bacterial

strains with transferrable resistant genes.

ACknowledgeMenTS

We thank Matthew Quattrini for his expert techni-

cal assistance.

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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 121

www.afabjournal.comCopyright © 2012

Agriculture, Food and Analytical Bacteriology

ABSTRACT

The objective of this study was to compare the bark and wood of low, non-irrigated, and high, irrigated,

specific gravity poplar clones for overall sugar recovery and sugar-degradation inhibitory byproducts

production when pretreated with dilute acid (160°C for 60 min in unstirred batch stainless steel reactors),

coupled to enzymatic hydrolysis. Overall, the combined xylose and glucose recoveries for low and high

specific gravity bark were 18.65 and 24.82 (g of sugar per 100 g of biomass), respectively, and 37.04 and

35.53 (g of sugar per 100 g of biomass) for low and high specific gravity wood, respectively. Total sugar

yields of 62 and 57% were calculated for low and high specific gravity wood, while sugar yields of 49 and

71% were obtained for low and high specific gravity bark. The glucose recovery was 48% for high specific

gravity wood and 55% for low specific gravity wood. The combined glucose and xylose content of low and

high specific gravity poplar wood was similar; yet, the glucose recovery in low specific gravity wood was

higher by 7%. The average ratio of sugar-derived inhibitory byproducts to potential sugars in high and low

specific gravity wood was 0.34; a similar value for bark was 0.66. Given the negative effects of inhibitory

byproducts on the bioprocessing chain, it may be prudent to omit bark when saccharifying poplar. The

low specific gravity clone displayed two advantages: 1) higher glucose recovery; and, 2) the ability to be

cultivated under dry land conditions. In summary, low specific gravity poplar clones, with rainfall as the sole

water supply, could prove to be viable feedstock sources.

Keywords: Poplar, dilute acid pretreatment, enzymatic hydrolysis, xylose, glucose, furfural, hydroxymeth-ylfurfural, formic acid, and acetic acid.

Correspondence: D. J. Carrier [email protected]: 479-575-2542; Fax: 479-575-2689

Sugar Recovery from the Pretreatment/Enzymatic Hydrolysis of High and Low Specific Gravity Poplar Clones

A. C. Djioleu1, A. Arora2, E. M. Martin1, J. A. Smith1, M. H. Pelkki3, and D. J. Carrier1

1Department of Biological and Agricultural Engineering, University of Arkansas, 203 Engineering Hall, Fayetteville, AR 72701

2Division of Microbiology, Indian Agricultural Research Institute, New Delhi, India 1100123School of Forest Resources, University of Arkansas at Monticello, Monticello, AR 71656

Agric. Food Anal. Bacteriol. 2:121-131, 2012

122 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

InTRoduCTIon

The development of second generation bioetha-

nol from lignocellulosic biomass shows significant

promise for the conservation of petroleum crude

and movement toward energy security. Cellulose

and hemicellulose, the most abundant constituents

of lignocellulosic biomass can be converted into fer-

mentable sugars for the production of ethanol. Dif-

ferent forestry products and residues, agricultural

byproducts, grasses, food processing and munici-

pal solid wastes can serve as lignocellulosic feed-

stock. Eastern cottonwood (Populus deltoides) is a

prevalent understory species in the southeast United

States. It is fast-growing, drought resistant, and has

the potential for becoming an economically valuable

feedstock for the production of cellulosic biofuels

(Kim et al., 2009; Sannigrahi and Ragauskas, 2010).

Sannigrahi and Ragauskas (2010) report that pop-

lar wood contains 39 to 49% (average 44%) glucan,

13 to 19% (average 15%) xylan and 17 to 29% (aver-

age 24%) lignin, on a dry weight basis. The pretreat-

ment of lignocellulosic biomass and subsequent en-

zymatic hydrolysis of cellulose to fermentable sugars

are the most cost intensive steps in converting bio-

mass to ethanol. Among the existing pretreatment

technologies, dilute acid pretreatment is preferred

since it removes a large fraction of the xylan and

opens pores for subsequent enzymatic hydrolysis of

the cellulose (Sannigrahi et al., 2011).

Martin et al. (2011) reported on the recovery of

xylose, the main component of hemicellulose, from

dilute acid pretreated high and low specific gravity

poplar clones. Pretreating in 1% dilute acid for 100

min in non-stirred reactors resulted in xylose recov-

eries of 55 and 50% for low and high-density poplar

clones. The low specific gravity clone was cultivated

under dry land conditions. These findings are im-

portant both environmentally and economically be-

cause not only would the omission of irrigation save

water, but a lack of irrigation would also reduce the

cost of transporting and/or pumping water to plant-

ing locations. This study is a continuation of past

research to further evaluate the saccharification ef-

ficiency of high and low specific gravity hybrid pop-

lar wood. The goal of this work is to maximize the

yield of hemicellulosic sugar, xylose, from dilute acid

pretreatment, and to link pretreatment to enzymatic

hydrolysis to yield the cellulosic sugar, glucose. In

addition, a mass balance of the monomeric sugars,

including sugar-degraded inhibitory byproducts,

was prepared. An analysis of these data was used

to determine differences in sugar recovery from the

high and low specific gravity clones, and to couple

the information with the decrease in sugar recovery

due to the production of degradation compounds

caused by the detrimental effects of dilute acid pre-

treatment.

MATeRIAlS And MeThodS

Poplar feedstock

The cottonwood clones were from Eastern Texas.

The clones were grown at the University of Arkansas

Pine Tree Branch Station; the 14 year-old mid-rota-

tion stands were harvested and used for this study.

The high specific gravity clones, S13C20, were irri-

gated during the first 10 yr growth and had a spe-

cific gravity of 0.48. The low specific gravity clones,

S7C15, were not irrigated and had a specific gravity

of 0.40.

Percent moisture/solid determination

Wood chips were ground to a 20 mesh particle size

using a Wiley Mini Mill (Torget et al., 1988). Ground

samples (approximately 0.5 g) were analyzed for per-

cent moisture/solid content using an Ohaus MB45

Moisture Analyzer (Parsippany, NJ).

Pretreatment

In preparing the biomass, one gram of 20 mesh

material and 20 mL of 0.98% H2SO4 were added to

thick-walled stainless steel reactors, (interior diam-

eter 14.22 mm, wall thickness 5.59 mm, length 200

mm, for a total chamber volume capacity of 32 ml)

and then placed in a fluidized sand bath (Techne In-

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 123

corporated, Burlington, NJ) at 160 °C for 60 min with

an air flow of 3.0 cubic foot per min.

The volume of the resulting hydrolysate was mea-

sured and recorded for quantification. Samples were

filtered through #1 Whatman filter paper, using a

Buchner funnel. Liquid fractions were analyzed by

high pressure liquid chromatography (HPLC) for the

presence of degradation compounds such as: fur-

fural, hydroxmethylfurfural (HMF), formic acid and

acetic acid prior to neutralizing. After neutralizing

with calcium carbonate, an aliquot of the liquid frac-

tion was analyzed by HPLC for carbohydrates. To

aid the enzymatic hydrolysis, the recovered solids

were washed with 30 mL of Millipore water, stirred

for 30 min, and filtered (Balan et al., 2009; Kumar and

Wyman, 2009). Liquid fractions from filtration were

analyzed by HPLC for both carbohydrates and inhibi-

tory compounds.

Enzymatic hydrolysis

To prepare for enzymatic hydrolysis, one gram of

solids, 500 µL of Accellerase ®1500 (Genencor) as

specified by the manufacturer, 5 mL of citrate buf-

fer, pH 4.8, and up to 10 mL of Millipore water were

added to 50 mL amber bottles. The bottles were

placed in a 55 °C shaking water bath for 48 h. Af-

ter completion of enzymatic hydrolysis, the samples

were boiled to denature the enzymes; the pH was

adjusted to neutral, and the samples were analyzed

for carbohydrate content.

Compositional Analysis

Procedure for compositional analysis of the bio-

mass followed those established in the technical re-

ports from the National Renewable Energy Labora-

tory (NREL) (Sluiter et al., 2008ab; Sluiter et al., 2011).

Poplar biomass, either wood or bark, was dried in a

120°C oven overnight prior to analysis.

HPLC analysis

Liquid aliquots from pretreatment, enzymatic

hydrolysis, and compositional analysis were fil-

tered through a 0.2 µm syringe filter, and analyzed

for carbohydrate content with a Waters 2695 HPLC

equipped with a Shodex SP-G pre-column and

SP0810 column (heated to 85 ºC) with water as the

eluent at a flow rate of 0.2 mL/min. Carbohydrates

were detected with a Waters 2414 Refractive Index

Detector. This method was adapted from National

Renewable Energy Laboratory (Sluiter et al., 2009a).

In detecting inhibitory compounds, liquid fractions

(that had not been neutralized) were filtered through

a 0.2 µm syringe filter and analyzed by Waters Al-

liance 2695 Separation Module, equipped with an

Aminex HPX-87H ion exchange column (300 mm X

7.8 mm), heated at 55 °C. Samples were run at a flow

rate of 0.6 mL/min and compounds were detected

by Waters 2996 Photodiode Array Detector at 280

nm for furfural and HMF and 210 nm for formic acid

and acetic acid.

Statistical Analysis

Calculations of carbohydrate and degradation

compounds (HMF, furfural, formic acid, and acetic

acid) were obtained using Microsoft Office Excel

2007. Analysis of the variance (ANOVA) was deter-

mined using JMP 9.0, LSMeans Differences Student’s

t, with α= 0.050.

ReSulTS And dISCuSSIon

The compositional analysis of oven-dried high

and low specific gravity bark and wood is presented

in Table 1. The percent solids for the compositional

analysis were 96% and 97%, respectively, for high and

low gravity poplar wood. Extractive concentrations

were similar for low and high specific gravity bark;

and for low and high specific gravity wood. The aver-

age xylose content was 12.3% for high specific grav-

ity wood and 13.3% for low specific gravity wood.

The average content was not significantly different

for the two wood densities. The average glucose

content was 46.3% for low specific gravity wood, and

49.7% for high specific gravity wood. Overall, these

values are similar to values previously reported by

124 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

Table 1. Compositional analysis of wood and bark from low and high specific gravity poplar

Compositional Analysis, g per 100 g of Biomass

Biomass Glucose Xylose Extractives Lignin/Ash Ash

Low wood 46.3 ± 2.57 13.3 ± 1.08 1.24 ± 0.26 18.48 ± 1.37 0.58 ± 0.13

High wood 49.7 ± 0.95 12.3 ± 0.26 1.48 ± 0.27 16.44 ± 1.73 0.37 ± 0.06

Low bark 25.8 ± 1.07 12.3 ± 0.87 8.58 ± 0.00 34.71 ± 2.46 4.20 ± 0.06

High bark 20.8 ± 2.44 14.2 ± 2.89 6.60 ± 0.00 37.89 ± 2.49 4.64 ± 0.39

Figure 1. Glucose and xylose recovery based on compositional analysis of low and high specific gravity wood (A) and low and high specific gravity bark (B) after dilute acid pretreatment and en-zymatic hydrolysis

0

20

40

60

80

100

120

Glucose Xylose

Pe

rce

nt

Re

cove

ry

Low wood

High wood

A

0

20

40

60

80

100

120

Glucose Xylose

Pe

rce

nt

Re

cove

ry

Low bark

High bark

B

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 125

Kim et al. (2009), Min et al. (2011), Sannigrahi et al.

(2010), and the United States Department of Energy

(2010). The bark composition was very different than

the composition of wood. Bark had on average ap-

proximately 25 g per 100 g of biomass less glucose

than that of wood. The low specific gravity clone’s

bark was thick and rough, in comparison to the high

specific gravity clone’s bark that was thinner and

smooth.

Martin et al. (2011) reported that dilute acid pre-

treatment of wood at 140ºC from the lower specific

gravity clone yielded the highest average xylose re-

covery of 56%. In this work, dilute acid pretreatment

of high specific gravity and of low specific gravity

clones at 140ºC and 160ºC resulted in xylose recov-

eries of 49 and 45%, respectively, which is lower than

previously reported. Martin et al. (2011) did not in-

tegrate pretreatment and enzymatic hydrolysis, and

overall carbohydrate recoveries were not calculated.

Moreover, carbohydrate yields were based on base-

line composition data provided by Kim et al. (2009).

In the present study, the pretreatment and hydrolysis

steps were integrated and calculations were based

on composition values presented in Table 1.

Of the pretreatment conditions explored by Mar-

tin et al. (2011), it was determined that the optimum

time/temperature parameters for dilute acid pre-

treatment, allowing for maximum glucose recovery

in subsequent enzymatic hydrolysis of poplar wood,

were 160°C for 60 min in 1% dilute acid. Xylose and

glucose recoveries from low and high specific grav-

ity poplar wood and bark are presented in Figure 1.

Monomeric sugar recovery was based on dry weight

percentages of the theoretical compositional analy-

sis (average percent solid of high specific gravity

wood and low specific gravity wood prior to pretreat-

ment was taken as 90%). Relating this to the percent

recovery after dilute acid pretreatment and enzymat-

ic hydrolysis, the xylose recovery was approximately

96% for high specific gravity and 87% for low spe-

cific gravity wood. The glucose recovery was 48% for

high specific gravity wood, and 55% for low specific

gravity wood. Results from this work indicated that

glucose yields from low specific gravity wood were

significantly higher than from high specific gravity

wood. Glucose recovery was adversely affected by

the high percentages of degradation products, par-

ticularly formic acid. Formic acid composition in low

specific gravity wood pretreatment hydrolysates was

as high as 15%, and approximately 14% in high spe-

cific gravity wood pretreatment hydrolysates.

Martin et al. (2011) also reported that the highest

xylose recovery from the dilute acid pretreatment of

low specific gravity bark was 31%, observed at 160°C

for 60 min. In varying the pretreatment temperatures

between 140°C and 200°C resulted in recoveries be-

tween 10 and 28%. The present work reports that di-

lute acid pretreatment at 160°C for 60 min, coupled

to enzymatic hydrolysis, resulted in higher overall

xylose and glucose recoveries. Specifically, the over-

all glucose and xylose recoveries were 37 and 73%,

respectively, for low specific gravity bark and 62 and

Table 2. Carbohydrate recovery from wood and bark in low and high specific gravity poplar

Recovery of Sugars, g per 100 g of Biomass

Pretreatment/Wash Enzymatic HydrolysisTotal

Glucose Xylose Glucose Xylose

Low Wood 3.53±0.20 11.35±1.85 21.95±1.42 0.21±0.02 37.04

High Wood 4.01±0.22 11.73±0.31 19.72±0.98 0.07±0.03 35.53

Low Bark 6.21±1.00 7.18±2.26 3.43±0.83 1.83±1.00 18.65

High Bark 6.57±0.73 8.94±3.02 6.40±0.55 2.91±0.89 24.82

126 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

84%, respectively, for high specific gravity bark. Thus,

as presented in Table 2, the combined xylose and

glucose recoveries for low specific gravity and high

specific gravity bark were 18.65 and 24.82 (g of sug-

ars per 100 g of biomass), respectively; and, 37.04

and 35.53 (g of sugars per 100 g of biomass) for low

and high specific gravity wood, respectively.

Poplar is touted as a potentially important woody

energy crop, and many groups have reported sugar

recovery from this feedstock. Negro et al. (2003),

using steam explosion pretreatment at 210°C for 4

min coupled to enzymatic hydrolysis, reported re-

coveries of 60 and 41% for glucose and xylose, re-

spectively. Pan et al. (2006, 2007) investigated the

conversion of a commercially available poplar clone

NM-6, Populus nigra X Populus maximowiczii, using

ethanol organosolv pretreatment with hot aqueous

ethanol. This process was implemented to not only

recover usable sugars for fermentation to ethanol,

but also to obtain a range of valuable lignin based

co-products. At 60 min 180°C pretreatment with

1.25% sulfuric acid and 60% ethanol, the reported

yields were 82% for glucose and 72% for xylose,

based on initial biomass composition. The effect of

various pretreatment technologies combined with

different plant cell wall hydrolyzing enzyme concen-

trations was reported by the Consortium for Applied

Fundamentals and Innovation (CAFI), sharing iden-

tical characterized poplar feedstock, thus, enabling

the comparison of processing technologies. The

series of papers were reported in one single 2009

issue of Biotechnology Progress. Balan et al. (2009)

reported glucan recoveries of 93% and xylan recov-

eries of 65%, when using ammonia fiber expansion

(AFEX) pretreatment coupled to enzymatic hydro-

lysis. Pretreatment conditions for optimum results

were a temperature of 180°C, 2:1 ammonia to bio-

mass loading, 23% moisture for a 30 min treatment,

with biomass milled to 50 mm. Using steam pretreat-

ment at 200°C, for 5 min with 3% SO2, Bura et al.

(2009) recovered 100% of the glucose and 89% of the

xylan. Kumar and Wyman (2009) compared a number

of the saccharification conditions and maximum xy-

lose recoveries using AFEX, ammonia recycled per-

colation (ARP), dilute acid, lime, controlled pH, and

sulfur dioxide (SO2) pretreatments also, linked to en-

zymatic hydrolysis. The maximum xylose recoveries

were 72.3, 94.6, 70.9, 72.9, 105.5 and 85.3 %, respec-

tively for the listed pretreatment techniques. Simi-

larly, Kumar and Wyman (2009) reported maximum

glucose recoveries of 61.1, 77.0, 89.8, 73.1, 65.9 and

90.8 %, respectively for AFEX, ammonia recycled

percolation (ARP), dilute acid, lime, controlled pH

and sulfur dioxide (SO2) pretreatments linked to en-

zymatic hydrolysis. Importantly, the work of Kumar

and Wyman (2009) reported that for the above list

of leading pretreatments, the maximum sugar yields

were 64.0, 79.6, 84.9, 71.9, 76.1 and 89.4 %, respec-

tively, indicating that SO2 pretreatment maximized

sugar recovery. In this work, total sugar yields of 62

and 57% were calculated for low and high specific

gravity wood, while sugar yields of 49 and 71% were

determined for low and high specific gravity bark.

Results reported in this work showed that the calcu-

lated recovery is similar to what was obtained with

AFEX, but lower than what can be derived from di-

lute acid and SO2 pretreatments (Kumar and Wyman,

2009). However, this work shows that more sugars

are recovered from low specific gravity poplar.

As illustrated by the CAFI work, there are a num-

ber of available pretreatment technologies; howev-

er, the dilute acid pretreatment, although it contains

many drawbacks, is more than likely to be adopted

at the deployment scale because of its low cost and

ease of use (Sannigrahi et al., 2011). Dilute acid pre-

treatment has been used for hardwoods (Esteghla-

lian et al., 1997; Jung et al., 2010; Lloyd and Wyman,

2005; Lu et al., 2009; Martin et al., 2010; Martin et al.,

2011; Schell et al., 2003; Torget et al. 1998; Wyman et

al., 2005; Wyman et al., 2008). However, dilute acid

pretreatment, which involves the use of high temper-

atures, is a harsh treatment that produces inhibitory

byproducts, including acetic acid, furfural, hydroxy-

methylfurfural (HMF), and formic acid (Palmqvist and

Hahn-Hagerdal, 2000). These degradation chemicals

reduce the sugar yields as well as ethanol yields by

50 to 60% (Du et al., 2010). Shuai, et al. (2010) com-

pared dilute acid pretreatment to sulfur dioxide-cat-

alyzed steam explosion (SPROL), using the softwood,

spruce, as the biomass feedstock. Known inhibitors

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 127

Figure 2. Pretreatment hydrolysate from low specific gravity wood (A) and low specific gravity bark (B). UV Chromatogram was acquired at 210 nm. The retention times of formic acid, acetic acid, HMF and furfural were approximately 13, 15, 30 and 44 min, respectively

A

B

128 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

were 35% lower with SPORL treatment compared

to dilute acid treatment. However, SPORL pretreat-

ments are costly and difficult to scale-up, further pro-

moting the use of dilute acid pretreatment.

Xylose is degraded into furfural, and further into

formic acid, while glucose is degraded into HMF, and

further degraded into formic acid and levulinic acid

(Palmqvist and Hahn-Hagerdal, 2000). In addition,

mannose and galactose go through similar degra-

dation pathways as glucose (Palmqvist and Hahn-

Hagerdal, 2000). Therefore, the yields of formic acid

during dilute acid pretreatment would increase not

only from degradation of xylose and glucose, but

also from the degradation of mannose and galac-

tose. Figure 2 presents a typical chromatogram from

wood and bark dilute acid hydrolysates, where for-

mic acid, acetic acid, HMF and furfural had retention

times of 13, 15, 30 and 44 min, respectively. Unfortu-

nately, many of the compounds seen in the UV trace

in Figure 2 remain unidentified. The intensity of the

unidentified 6.1 min peak in the bark UV trace is over

1 absorbance units, dwarfing all of the other peaks in

the chromatogram. Moreover, the work of Du et al.

(2010) illustrates that a UV trace does not detect all

of the compounds that are present. While compar-

ing the chromatograms of dilute acid pretreatment

corn stover prehydrolysates, Du et al. (2010) detect-

ed 38 compounds when using mass spectrometry

and only two compounds with UV detection. Thus,

the chromatograms presented in Figure 2 may not

fully illustrate the entire slate of compounds present

in poplar dilute acid hydrolysates.

Table 3 represents the calculated yields, of in-

hibitory products (g per 100 g of biomass) that were

generated from wood and bark when pretreating

with dilute acid. The values in Table 3 values were

calculated by tying the concentrations of inhibitors

in the prehydrolysates (determined by HPLC) to the

volume of hydrolysate and masses of the pretreated

feedstock. Of the four monitored inhibitory prod-

ucts, formic acid displayed the highest concentra-

tion, ranging from 14 to 15 g per 100 g of hydrolyzed

biomass. Formic acid is a known enzymatic hydrolysis

inhibitor (Cantarella et al., 2004). Hodge et al. (2008)

illustrated unequivocally that if inhibitory products,

such as formic acid, were not removed from the pre-

treated biomass, the reactor could not be loaded

with more than 15% (v/v). By washing the pretreated

biomass with three volumes of water, to remove the

inhibitory products from the pretreated corn stover

biomass, Hodge et al. (2008) showed that the reactor

could be loaded to 30% (v/v), illustrating the impor-

tance of removing or minimizing inhibitory products.

Similarly, Kumar and Wyman (2009) reported that

the enzymatic hydrolysis step could be enhanced by

10% when washing the dilute acid pretreated bio-

mass prior to treatment with enzymes. In this work,

the ratio of inhibitory compounds (Table 3) to po-

tential sugars in wood and bark (Table 2) was 0.34

and 0.66 (g per g), respectively. Given the negative

Table 3. Inhibitory products detected in low and high specific gravity poplar wood and bark di-lute acid pre-hydrolysates

  Inhibitors in Prehydrolysates, g per 100 g of Biomass

Biomass HMF Furfural Formic acid Acetic acid

Low wood 0.13±0.24 1.90±0.21 15.01±2.67 4.46±0.92

High wood 0.12±0.00 1.72±0.06 13.59±0.18 4.88±0.05

Low bark 0.46±0.11 2.66±0.64 14.08±1.62 5.21±0.76

High bark 0.54±0.12 2.49±0.51 16.71±1.86 6.17±0.60

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 129

effects of the inhibitory compounds on the biopro-

cessing chain, as highlighted by Hodge et al. (2008)

and Kumar and Wyman (2009), it may be prudent to

omit bark when saccharifying poplar.

From a sustainability perspective, growing feed-

stock with minimum amounts of water can be a sub-

stantial advantage. The low specific gravity poplar

was cultivated in southern Arkansas for 15 years, us-

ing rainfall as the sole water supply. The data pre-

sented in this study highlight, in fact, that the low

specific gravity clone yields 4% more carbohydrates

than its high density irrigated counterpart. This in-

creased carbohydrate yield could offset the greater

growth observed in irrigated clones and will play a

role in determining the total cost of carbohydrate

production from poplar plantations. Data from this

present study illustrate that non-irrigated low spe-

cific gravity clones displayed comparable feedstock

characteristics to those of irrigated high specific

gravity clones. The study presented by Negro et al.

(2003) was also concerned with water usage, and

stated that Populus nigra is promising for south and

central Europe, due to the high yield and drought

resistance of this species.

ConCluSIonS

This study reports that non-irrigated 15 year-old

low specific gravity poplar wood yielded compa-

rable percentages of xylose, but significantly higher

glucose percentage recovery, when pretreated with

dilute acid coupled to enzymatic hydrolysis than its

irrigated high specific gravity counterpart. Although

this is a preliminary study, specific gravity may be an

important property to predict the saccharification

potential of poplar biomass.

ACknowledgeMenTS

The authors would like to thank the University of

Arkansas, Division of Agriculture, and the Depart-

ment of Biological and Agricultural Engineering, for

financial assistance. The authors would also like to

acknowledge National Science Foundation award

no. 0828875; Department of Energy award no.

08GO88035 for the pretreatment equipment and

support of EMM; and CRREES National Research

Initiative award no. 2008-01499 for the HPLC instru-

ment. The authors are very grateful to Dr. Ed Clau-

sen for critically reviewing this manuscript.

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132 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

www.afabjournal.comCopyright © 2012

Agriculture, Food and Analytical Bacteriology

ABSTRACT

Microbial diversity in tannery waste disposal sites was characterized in this research. The research work

consisted of isolation and cultivation of microbes on laboratory media and the subsequent characteriza-

tion of pure isolates. The 16S rRNA gene of isolated bacteria was analyzed for molecular characteriza-

tion of bacterial diversity at those areas. 16S rRNA gene amplification was successful for sixteen isolates.

Restriction digestion of these amplified 16S rRNA genes by two different restriction enzymes, HinP1I and

MspI, revealed six different restriction patterns of the 16S rRNA gene. Representative 16S rRNA genes of

these six different patterns were selected for DNA sequencing to characterize these bacteria further. 16S

rRNA genes of five of the representative bacterial colonies of the six selected patterns were successfully

sequenced. The identified bacterial species included both gram positive and gram negative bacteria from

the groups γ-proteobacteria and Firmicutes.

Keywords: Hazaribagh, Tannery, Bangladesh, Molecular diversity

InTRoduCTIon

Leather, a traditional export item in Bangladesh,

enjoys a good reputation worldwide for its quality

(Sharif and Mainuddin, 2003). This sector plays a sig-

nificant role in the economy of Bangladesh in terms

of its contribution to export and domestic market

(Sharif and Mainuddin, 2003). In Bangladesh, tanning

Correspondence: S. Yeasmin, [email protected]; [email protected]: +1 -229-386-3363 Fax: +1-229-86-3239

or the process of making leather is mostly carried out

in the south-western region of Dhaka city, occupying

25 hectares of land at Hazaribagh, where about 90%

of tannery industries of Bangladesh are located. Due

to lack of appropriate waste management practices,

both solid wastes and liquid effluents from these

industries are deposited at different low-lying loca-

tions of Hazaribag without proper treatments (Zahid

et al., 2006). Components of these wastes include

rotten flesh, fat, blood and skin, toxic chemicals,

dissolved lime, chromium sulfate, alkali, hydrogen

Culture dependent molecular analysis of bacterial community of Hazaribagh tannery exposed area in Bangladesh

A. A. Maruf 1, 3, 5, M. M. Moosa1, 4, 5, S. M. M. Rashid 1,5, H. Khan2, S. Yeasmin1

1 Department of Genetic Engineering and Biotechnology, University of Dhaka, Dhaka 1000, Bangladesh.2 Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka 1000, Bangladesh.

3 Present address: UChicago Research Bangladesh Ltd., House 338, Road 24, New DHOS, Mohakhali, Dhaka 1206, Bangladesh.

4 Present address: Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka 1000, Bangladesh.5 Contributed equally to the study.

Agric. Food Anal. Bacteriol. 2:132-138, 2012

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 133

sulfide, sulfuric acid, bleach, dyes, oil, formic acid,

heavy metals, suspended solids, organic matters

and so on (Bhuiyan et al., 2011; Zahid et al., 2006).

Consequently, soil sediment, groundwater and sur-

face water of nearby rivers (Buriganga and Turag) are

polluted heavily through percolation of the leachate

from these dumping sites (Zahid et al., 2006).

Despite the toxic chemical load, a number of bac-

terial species are found to be abundant in the micro-

flora of tannery effluents (Lefebvre et al., 2006; Tripa-

thi et al., 2011). While the details of the mechanism

of resistance of this microflora to toxic chemicals are

yet to be deciphered, three possible mechanisms

are likely to operate separately or in combination:

a) efflux systems can reduce toxic chemical loads

(Mosqueda and Ramos, 2000; Nies and Silver, 1989;

Nies and Silver, 1995); b) toxic chemicals can be de-

graded (Franco et al., 2005) or converted to less tox-

ic forms (Basu et al., 1997; Fakruddin et al., 2009; Ilias

et al., 2011; Masood and Malik, 2011; Rafiqullah et

al., 2009); c) toxic chemicals can be sequestered into

complex compounds (Nies, 1999) or compartments

(Avery, 1995; Cooksey, 1993). The study of these

microorganisms and/or their mode of resistance can

lead to the development of novel biological meth-

ods of remediation of these toxic chemicals which

can be less expensive compared to physicochemical

remedial strategies (Farabegoli et al., 2004; Hasega-

wa et al., 2010; Sivaprakasam et al., 2008; Tripathi et

al., 2011; Yan et al., 2011). This study aimed to char-

acterize microbial contents of tannery effluent en-

riched environment of Hazaribagh region of Dhaka.

MATeRIAlS And MeThodS

Sample collection

Samples were collected both from long-term and

short time tannery waste disposal sites of Hazaribag

tannery area to ensure proper representation of mi-

crobial community diversity. The sites of sample col-

lection was categorized into four broad categories: a)

short term tannery waste disposal site; b) long term

tannery waste disposal site; c) submerged tannery

waste disposal site and d) edges of tannery waste

disposal pond.

Sample preparation

A 5 g waste sample was mixed with 30 mL distilled

water. It was divided into two 15 mL test tube and

centrifuged at 2000 rpm for five minutes. A volume of

100 µL supernatant from each sample was inoculated

on lysogeny broth (LB) agar plate and incubated at

37°C.

Pure culture isolation

Pure cultures were isolated from the mixed cul-

tures of microorganisms. The pure cultures were pre-

pared by streak plate method and preserved at 4°C

in a refrigerator. Sampling was done twice from the

sites in January, 2009 and March, 2009. A total of 28

colonies were selected based on colony morphology

and labeled as SY-1 to SY-28. These 28 isolates were

studied further.

Isolation of genomic DNA from bacteria

Colonies from pure cultures were inoculated into

LB media. The cultures were incubated overnight

at 37°C temperature at 120 rpm. A 1.5 mL bacterial

culture aliquot was placed into a sterile Eppendorf.

It was spun in a centrifuge at 10,000 rpm for 2 min-

utes. Later the supernatants were discarded and

bacterial cells were harvested. Genomic DNA was

isolated from these bacterial cells according to the

CTAB (Cetyl Trimethyl Ammonium Bromide) method

(Chen and Kuo, 1993) with slight modifications. Solu-

tions of 567µL TE (Tris EDTA), 3 µL 10% SDS and 3µL

of 20 mg/ml proteinase K were added with the cell

pellets and thoroughly mixed. The suspension was

then incubated for 1 hour at 37°C in water bath. A 5M

NaCl (100µL) solution was added to this suspension

and mixed thoroughly. An 80µL CTAB/NaCl solution

was added, mixed thoroughly and the resulting sus-

pension was incubated at 65°C. Equal volumes of

chloroform: Isoamyl Alcohol (IAA) equivalent to 24:1

were added to the suspension and shaken vigorously

134 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

for 15 minutes. The resulting suspension was spun at

14000 rpm for 10 minutes. The aqueous phase from

the Eppendorf tube was transferred to a new steril-

ized Eppendorf tube.

Equal volumes of Phenol: Chloroform: IAA (Iso-

amyl Alcohol) = 25: 24: 1 were added to it and mixed

thoroughly by inverting the tube for five minutes.

The resulting suspension was spun at 14,000 rpm

for 10 minutes. The aqueous phase (above the white

interface layer) was transferred carefully by a 200 µL

micropipette to a clean tube and the remainder was

discarded. DNA was precipitated with double vol-

ume of absolute ethanol. It was spun for 15 minutes

at 14,000 rpm at 4°C to pellet the DNA. The pellet

was collected and washed once or twice with ice

cold EtOH. It was done once with 70% EtOH or 76%

EtOH/10 mM AcNH4 (ammonium acetate). A second

wash with 70% EtOH was subsequently performed.

It was spun 15 min at 14,000 rpm, 4°C. The superna-

tant was removed and the pellet was dried by leaving

tube open at room temperature for an hour. The pel-

let was resuspended in TE (pH of TE was maintained

8.0). The aliquot of pellet was stored at -20°C.

PCR amplification of 16s rRNA gene

Isolated bacterial genomic DNA was used as

template to amplify 16S Small-subunit (SSU) rRNA

genes by PCR using the primers 8F (5’-AGAGTTT-

GATCCTGGCTCAG) and 805R (5’-GACTACCAGGG-

TATCTAAT) as described by Lane and coworkers

(Lane et al., 1985). Each 25µL PCR mixture included

2.5 µL of 10X PCR buffer, 2 µL of deoxynucleoside tri-

phosphate mix, 1.5 µL of MgCl2, 0.5µL of each primer,

0.3 µL of Taq polymerase (Takara Taq™, Otsu, Japan),

1 µL of genomic DNA lysate and 16.7 µL of PCR H2O

(following the manufacturers’ protocols). Thirty cycles

of amplification (92°C for 30 s, 58°C for 40 s, and 72°C

for 90 s) usually were sufficient to obtain a product of

the appropriate length that was visible in ethidium

bromide-stained agarose gels.

Restriction Fragment Length Polymor-phism and sequencing

The PCR product was digested with 2 U each of

MspI (Takara, Japan) and HinP1I (Takara, Japan) re-

striction enzymes for 1 h at 37°C. Each restriction

digestion mixture contained 7 µL PCR product, 2 µL

10x T Buffer, 2 µL 0.1% BSA, 2 µL respective restric-

tion enzyme and the final volume was brought to 23

µL by adding 10 µL nuclease free H2O. Besides, PCR

product was digested with both these restriction

enzymes. The restriction fragments were separated

by gel electrophoresis on 2% Gene-Pure agarose

(Sigma). Restriction fragment length polymorphism

(RFLP) types were sorted by visual inspection of digi-

tized gel images. Representative RFLP types were

sequenced from Macrogen Inc. (Seoul, Republic of

Korea) using the reverse primer 805R.

Phylogenetic analysis

The evolutionary history was inferred using the

Minimum Evolution method (Rzhetsky and Nei,

1992). The percentage of replicate trees in which the

associated taxa clustered together in the bootstrap

test (10000 replicates) is shown next to the branch-

es (Felsenstein, 1985). The tree was drawn to scale,

with branch lengths in the same units as those of the

evolutionary distances used to infer the phylogenetic

tree. The evolutionary distances were computed us-

ing the Maximum Composite Likelihood method

(Tamura et al., 2004) and were in the units of the

number of base substitutions per site. The ME tree

was searched using the Close-Neighbor-Interchange

(CNI) algorithm (Nei and Tamura, 2000) at a search

level of 3. The Neighbor-joining algorithm (Saitou

and Nei, 1987) was used to generate the initial tree.

All positions containing gaps and missing data were

eliminated from the dataset (complete deletion op-

tion). There were a total of 556 positions in the fi-

nal dataset. Phylogenetic analyses were conducted

using the software MEGA4 (Tamura et al., 2007).

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 135

ReSulTS

Isolation of pure culture

A total of 28 pure cultures were isolated from four

different sampling categories based on unique mor-

phological appearance of the initial mixed culture.

They were labelled from SY-1 to SY-28. Three differ-

ent colonies (SY-12, SY-17 and SY-18) were collected

from long term waste disposal site, 5 different colo-

nies (SY-4, SY-5, SY-7, SY-8 and SY-21) from short term

waste disposal site, 9 different colonies (SY-2, SY-3,

SY-9, SY-15, SY-16, SY-22, SY-26, SY-27 and SY-28)

from submerged waste and 11 other colonies (SY-1,

SY-6, SY-10, SY-11, SY-13, SY-14, SY-19, SY-20, SY-23,

SY-24 and SY-25) from the edges of the waste dispos-

al pond. Of these 28 pure colonies PCR amplification

of 16S SSU rRNA was successful for the recovered

genomic DNA from 16 colonies. Of them, 4 colonies

failed to show any banding pattern upon restriction

digestion (SY-7, SY-12, SY-16 and SY-17). RFLP based

screening (MspI, HinP1I and MspI+HinP1I) of the re-

mainder of the 12 colonies identified six different re-

striction patterns. Representative colonies from each

pattern were selected (SY-9, SY-14, SY-23, SY-24, SY-

26 and SY-27) for DNA sequencing. The relationship

between different colonies and respective restriction

pattern is given in Table 1.

Identification of Bactria by 16S rRNA gene sequence comparison

Sequencing of the 16S ribosomal RNA gene was

successful for five bacterial strains (SY-14, SY-23, SY-

24, SY-26 and SY-27). Sequences were deposited

at the DDBJ/EMBL/GenBank nucleotide sequence

databases with accession numbers AB622992 to

AB622996. Sequencing of 16S rRNA gene of SY-9

was not successful. Nucleotide blast suggested

close homology of these sequences with different

soil bacterial species (Table 2). Phylogenetic analysis

suggested a close relationship between the identi-

fied microorganisms (Figure 1).

dISCuSSIon

Bioremediation by microorganisms is one of the

efficient and economical ways to decontaminate

sites of pollution. Exploring the microbial diversity

is the key to develop this effective and environmen-

tally friendly technology (Paul et al., 2005). Due to

lack of proper waste management practices, Haz-

aribag has become heavily polluted with effluents

from nearby tannery industries (Arias-Barreiro et al.,

2010; Bhuiyan et al., 2011; Zahid et al., 2006). How-

ever, little is known about the microbial flora of this

region and their interaction with the ecosystem. The

current study aimed to explore microbial diversity of

this area.

To ensure proper representation, samples were

collected from four different types of waste disposal

sites of Hazaribagh tanning area (short term tannery

waste disposal site; long term tannery waste disposal

site; submerged tannery waste disposal site; and the

edges of tannery waste disposal pond) as discussed

in the Materials and Methods section. Twenty eight

different pure culture colonies (SY-1 to SY-28) were

isolated from the collected samples.

Pure culture technique was followed to isolate the

differing colonies so that all of the individuals in a

Table 1. The relationship between different colonies and respective restriction pattern. Strains marked with bold numbers were selected for sequencing. SY-9 could not be sequenced.

Pattern Bacterial Strains

I SY-23

II SY-26

III SY-9

IV SY-24,1, 25

V SY-27, 28, 2

VI SY-14, 15, 4

136 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

culture have originated from a single individual. This

allowed the characterization of microorganisms with-

out confounding presence of other different types

of microorganisms. While isolating microorganisms

from complex mixtures, the isolation procedure was

repeated at least once to make sure that an isolat-

ed colony was single cell derived. These microbial

colonies were isolated by observation of colony mor-

phology. Samples collected from long term waste

disposal site showed the highest microbial diversity.

Genomic DNA was isolated from the pure cultures.

The amount of genomic DNA isolated was minimal

for some colonies but still sufficient to carry out am-

plification of the 16S rRNA gene.

Isolated genomic DNA was amplified with primers

specific for bacterial 16S rRNA gene (8F and 805R).

PCR was successful for 16 pure cultures (SY-1, SY-2,

SY-4, SY-7, SY-9, SY-12, SY-14 to 17, and SY-23 to 28).

16S rRNA genes of other 12 microbial colonies were

not amplified by polymerase chain reaction. Later, six

different restriction patterns were observed in these

16 PCR products by digesting the PCR products with

two different restriction enzymes HinP1I and MspI,

both alone and in combination. One colony from

each pattern was selected for 16S rRNA gene se-

quencing.

Sequencing was successful for five bacterial

strains. After 16S rRNA gene sequence analysis bac-

terial strains SY-14, SY-23, SY-24, SY-26 and SY-27 ex-

hibited the best nucleotide blast match with Provi-

dencia vermicola strain AR_PSBH1, Providencia sp.

FD32, Bacillus subtilis strain Bio AAS1, Bacillus sp.

FRC_Z41 and Proteus penneri strain Z2 respectively.

The identified bacterial species included both gram

positive and gram negative bacteria from the groups

γ-proteobacteria and Firmicutes (Table 2).

Table 2. Identification of Bacteria by 16S rRNA gene sequence comparison

Bacteria strain Accession The best BLASTn match Group

SY-14 AB622992Providencia vermicola strain AR_PSBH1

γ-proteobacteria Gram negative

SY-23 AB622993 Providencia sp. FD32 γ-proteobacteria Gram negative

SY-24 AB622994Bacillus subtilis strain Bio AAS1

Firmicutes Gram positive

SY-26 AB622995 Bacillus sp. FRC_Z41 Firmicutes Gram positive

SY-27 AB622996 Proteus penneri strain Z2 γ-proteobacteria Gram negative

Figure 1. Analysis of evolutionary relationship between the 5 taxa. Previously classified Providen-cia rettgeri (GenBank accession: FJ151630) and Bacillus tequilensis (GenBank accession: FR865173) was added to the tree to show γ- proteobacteria and Firmicutes specific clustering of the clades.

γ-proteobacteria

Firmicutes

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 137

A literature survey revealed that similar bacterial

species were previously identified in different stud-

ies on tannery effluent and soil microflora. Members

of Providencia genus are known to reside in different

soil habitats (Rani et al., 2008; Thacker et al., 2006).

Different species of Proteus (Usha and Kalaiselvi,

2009) and Bacillus (Chaturvedi, 2011; Megharaj et al.,

2003) genus were found in tannery effluents. Thus,

results of the study are in harmony with those of pre-

vious studies.

Considerable research has been performed

throughout the world to determine the microbial

diversity of different communities. Nevertheless, to

our knowledge, no study was carried out to evalu-

ate molecular microbial diversity of tannery exposed

area in Bangladesh. The current study was per-

formed with a view to shed light on this important

aspect. The study identified five different bacterial

species which were present in the tannery effluent

exposed area. Further biochemical characterization

of individual bacterial species could elucidate de-

tails of their mechanism of survival which can later

be used for bioremediation purposes.

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Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 139

www.afabjournal.comCopyright © 2012

Agriculture, Food and Analytical Bacteriology

ABSTRACT

The use of by-product feedstuffs and prebiotics in animal diets has increased in recent years. The pres-

ent study was undertaken to determine what effects novel by-product feedstuffs, including prebiotics, have

on survival of the important foodborne pathogenic bacteria Escherichia coli O157:H7 and Salmonella en-

terica Typhimurium in pure and mixed ruminal and fecal culture fermentations from cattle and swine. By-

product feedstuffs utilized in this study included: hyperimmunized whole egg, lysine biomass, lysine bio-

mass (spray dried), threonine biomass (drum dried), threonine biomass (spray dried), beer well yeast (drum

dried), beer well yeast (spray dried), ethanol yeast (pan dried) and corn meal as a control to simulate normal

dietary conditions. Prebiotics examined included: PremiDex ™, CitriStim™, a CitriStim:PremiDex blend

(50%:50%), and a commercial oligosaccharide source feedstuff. Pure culture populations of E. coli O157:H7

were reduced (P < 0.05) by 2% w/v of each of spray dried threonine, drum-dried threonine, ethanol yeast,

hyper-immunized whole egg, and a blend of CitriStim:PremiDex. No effects on Salmonella populations

were observed in pure cultures. Fermentations of mixed ruminal microorganisms from cattle fed a forage

based diet demonstrated that 2% PremiDex reduced (P < 0.05) E. coli O157:H7 populations compared

to controls i and the CitriStim:PremiDex blend reduced E. coli O157:H7 and Salmonella populations (P <

0.05) in fermentations from cattle fed high grain diets. The anti-foodborne pathogen effects appear to be

an indirect effect mediated by the microbial population of the intestinal tract, such as has been reported

previously for prebiotics.

Keywords: by-products, prebiotics, probiotic, feedstuff, E. coli O157:H7, Salmonella, food safety

Correspondence: T. R. Callaway, [email protected]: +1-979-260-9374 Fax: +1-979-260-9332

Impact of By-product Feedstuffs on Escherichia coli O157:H7 and Salmonella Typhimurium in Pure and Mixed Ruminal and Fecal Culture in Vitro†

T. R. Callaway1, S. Block2, K. J. Genovese1, R. C. Anderson1, R. B. Harvey1, D. J. Nisbet1

1USDA/ARS, Food and Feed Safety Research Unit, College Station, TX 778452Archer Daniels Midland Company, Decatur, IL

† Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies neither approval of the product,

nor exclusion of others that may be suitable. USDA is an equal opportunity provider and employer.

Agric. Food Anal. Bacteriol. 2:139-148, 2012

140 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

InTRoduCTIon

Enterohemorrhagic Escherichia coli (EHEC), such

as E. coli O157:H7, and Salmonella enterica are two

of the most critical foodborne pathogenic bacteria

and are often found asymptomatically in the gastro-

intestinal tract of farm animals (Ferens and Hovde,

2011; Scallan, et al., 2011). Both pathogens can sur-

vive undetected within the gastrointestinal micro-

bial population of cattle (Berg, et al., 2004; Callaway,

et al., 2006; Kunze, et al., 2008), and Salmonella is

often found in cattle, swine and poultry (Borland,

1975; Davies, et al., 1999). When these pathogens

are transmitted to a human they cause severe illness

or even death, the combined yearly cost of these

pathogens to the U.S. economy is in excess of $15

billion (Scharff, 2010).

Dietary effects on gastrointestinal pathogen pop-

ulations have been examined extensively, and the

effects have been extremely variable depending on

the specific feedstuff utilized and the production

situation (Callaway, et al., 2009; Diez-Gonzalez, et al.,

1998; Kudva, et al., 1995). In recent years there has

been a dramatic increase in the use of corn based

by-product feedstuffs in food animal diets (Richman,

2007). This growth has been especially notable in

the ethanol fermentation by products, such as distill-

ers grains (DG). However, research has shown that

the inclusion of DG in cattle diets can increase fecal

prevalence and shedding of E. coli O157:H7 (Jacob,

et al., 2009; Jacob, et al., 2010; Jacob, et al., 2008c;

Wells, et al., 2009). The present study was designed

to examine a variety of novel corn-derived feedstuffs

and selected commercially available prebiotic prod-

ucts (PremiDex, commercial oligosaccharide, CitriS-

tim) in regard to their ability to provide a selective ef-

fect on E. coli O157:H7 and Salmonella Typhimurium

populations in pure and mixed culture in vitro.

MATeRIAlS And MeThodS

Bacterial strains and culture conditions

Escherichia coli O157:H7 strain 933 (ATCC 43895)

was originally isolated from a human hemorrhagic

colitis outbreak, and the Salmonella enterica Ty-

phimurium isolates that were used in this study were

originally isolated from cattle and swine. All isolates

were obtained from the Food and Feed Safety Re-

search Unit (USDA/ARS, College Station, TX) culture

collection. Both E. coli O157:H7 strain 933 and S.

Typhimurium were selected for resistance to novo-

biocin (No) and nalidixic acid (NA; 20 and 25 µg/

mL, respectively) by repeated transfer and selec-

tion in the presence of sub-lethal concentrations of

each antibiotic. This resistant phenotype was stable

through multiple unselected transfers in batch cul-

ture and through repeated culture vessel turnovers

in continuous culture (data not shown).

Pure culture studies

Feedstuffs were added to 16 x 100 mm tubes

containing anoxic Trypic Soy Broth (TSB, (cooled

after autoclaving under anoxic conditions [90% N2,

5% H2, 5% CO2]; Difco Laboratories; Detroit, MI) to

reach final concentrations of 2% wt/vol. Feedstuffs

utilized in this study were: PremiDex, CitriStim, a

CitriStim:PremiDex, commercial oligosaccharide,

hyperimmunized whole egg, lysine biomass (drum

dried), lysine biomass (spray dried), threonine bio-

mass (drum dried), threonine biomass (spray dried),

beer well yeast (drum dried), beer well yeast (spray

dried), ethanol yeast (pan dried) and corn meal as a

control. Corn meal was used as a control to simu-

late the typical ingredients in a ruminant diet that

would be replaced by the by-product feedstuffs.

Feedstuffs were added into tubes immediately be-

fore the pathogens were inoculated. Escherichia coli

O157:H7 strain 933 (ATCC 43895) or S. Typhimurium

were grown anaerobically and aseptically inoculated

into feedstuff containing tubes at approximately 6 x

105 CFU/mL of E. coli O157:H7 or 3 x 105 CFU/mL of

S. Typhimurium at t = 0. Tubes in triplicate (n = 3)

were incubated at 39 °C for 24 h, when 1 mL aliquots

were removed for enumeration (described below).

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 141

Animal diets

All animals were maintained in accordance with a

protocol approved by the Southern Plains Agricul-

tural Research Center Animal Care and Use Commit-

tee (ACUC No 06002). Holstein cattle (n = 4) were

provided ad libitum access to water and minerals

at all times. Ruminal fluid was collected from cattle

grazing bermudagrass pasture (n=2) and a high-

grain diet (n=2). Cattle in the pasture-fed group

were grazed on an early vegetative stage ryegrass

pasture at the time of ruminal fluid collection. The

grain-fed cattle were fed a commercial feedlot ra-

tion (corn soybean mix; Producer’s Co-op, Bryan, TX)

and was maintained on this feedlot diet for 10 d prior

to ruminal or fecal collection. Crossbred pigs (n=4)

were provided ad libitum access to water and miner-

als at all times. Pigs were fed a commercial finish-

ing diet (Producer’s Co-op, Bryan, TX ) comprised of

soybean meal and corn twice daily. Animals were

maintained on this diet for 14 d prior to fecal fluid

collection.

Ruminal and fecal fluid collection

Ruminal contents were collected by hand from

the ventral sac of two ruminally cannulated Holstein

cows on each diet (n = 2/diet group). The ruminal

contents were collected from all cattle at approxi-

mately the same time (between 0800 and 0900 h).

Immediately after removal from the rumen, the con-

tents from each cow were strained via a fine mesh

nylon strainer (Reaves and Co., Durham, NC) and

pooled. Ruminal fluid was transported to the labo-

ratory and incubated for 30 min at 39 °C to allow gas

production to buoy large particles to the top of the

flasks. Fresh ruminal fluid contained approximately

1010 cells/ml of total culturable anaerobes, as deter-

mined by serial dilution in anaerobic reinforced clos-

tridial broth in triplicate tubes.

Fecal samples from cattle were collected directly

per rectum by digital grab. Feces were collected

from all cattle at the same time. Immediately upon

collection, the feces were strained via a fine mesh

nylon strainer to obtain a fecal fluid which was then

pooled. Fecal fluid was transported to the labora-

tory as described above. Fresh fecal fluid contained

approximately 1010 cells/ml of total culturable an-

aerobic bacteria as determined by serial dilution as

described above.

Swine feces were collected directly per rectum by

digital grab from all pigs at the same time, pooled

equally by weight, and was added directly to the fe-

cal fermentation media (described below) at a 33%

w/v final concentration. Fresh swine feces contained

approximately 109 cells/g of total culturable anaero-

bic bacteria.

In vitro mixed ruminal microbial fermen-tations

Incubations of cattle ruminal and fecal fluids were

performed by combining the gastrointestinal fluid

(33% vol/vol) with an anoxic basal medium contain-

ing (per liter): 292 mg K2HPO4, 202 mg KH2PO4, 436

mg NH4SO4, 480 mg NaCl, 100 mg MgSO4•7H2O, 64

mg CaCl2•H2O, 4,000 mg Na2CO3, 600 mg cysteine

hydrochloride (Cotta and Russell, 1982) supplement-

ed with 1 g/L glucose. Swine feces was added to

the same basal medium in a 33% w/v concentration.

Approximately 104-5 CFU/mL E. coli O157:H7 strain

933 or S. Typhimurium were added to the buffered

gastrointestinal fluid fermentations in all experi-

ments. The resultant suspensions were anaerobi-

cally transferred to 18 × 150 mm Balch tubes (Bellco

Glass, Vineland, NJ; 10 ml per tube). Feedstuffs de-

scribed above were added to each tube to reach fi-

nal concentrations of 2 % wt/vol under an anoxic gas

phase. Tubes in triplicate (n = 3) were then sealed

using rubber stoppers with aluminum crimps and in-

cubated for 24 h at 39°C under a N2, CO2, H2 (90:5:5

v/v) gas phase. Samples were removed after 24 h of

incubation and centrifuged (10,000 × g, 5 min, 24°C)

to remove particulate matter.

Bacterial enumeration

Samples were taken from all pure and mixed cul-

ture in vitro fermentations at 24 h to determine the

effect of feedstuffs on populations of E. coli O157:H7

142 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

and S. Typhimurium. Samples were serially diluted

(in 10-fold increments) in phosphate buffered saline

(PBS, pH 7.0), and subsequently plated on MacCo-

nkey’s agar (supplemented with 25 µg/mL NO and

20 µg/mL NA) and incubated at 37°C overnight for

direct counting of E. coli O157:H7 CFU/ml. To deter-

mine populations of S. Typhimurium, samples were

serially diluted as described above and plated on

Brilliant Green Agar (supplemented with 25 µg/mL

NO and 20 µg/mL NA) and incubated at 37°C over-

night for direct counting.

Statistical analysis

Pure culture experiments were performed with

(n=3) tubes on consecutive days. Mixed ruminal

bacteria experiments were performed in duplicate

tubes (n=2) on consecutive days, and the values pre-

sented are means. Students’ t-test was used to de-

termine significance of differences between means

of each treatment.

ReSulTS

In pure culture studies, none of the feedstuffs

used in this study affected Salmonella Typhimurium

populations (Figure 1) compared to controls using

corn meal. However, populations of E. coli O157:H7

in pure cultures were reduced (P < 0.05) when: spray

dried threonine biomass, drum-dried threonine bio-

mass, ethanol yeast, hyper-immunized whole egg,

and a blend of CitriStim:PremiDex were included;

although it must be noted that this statistically sig-

Figure 1. Effects of feedstuffs on populations of E. coli O157:H7 (light bars) and Salmonella Ty-phimurium (dark bars) in pure cultures. Feedstuffs were included at 2% w/v, and bars represent the mean of 3 fermentations, and error bars indicate standard deviation. Bars marked with superscript a indicate differences from control of P < 0.05.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 143

Figure 2. Effects of feedstuffs on populations of E. coli O157:H7 (light bars) and Salmonella Ty-phimurium when inoculated into mixed ruminal microorganism fermentations from cattle fed a forage-based diet. Feedstuffs were included at 2% w/v, and bars represent the mean of 3 fermen-tations, and error bars indicate standard deviation. Bars marked with superscript a indicate differ-ences from control of P < 0.05.

nificant reduction was less than a 10-fold decrease in

E. coli O157:H7 populations (Figure 1).

When pathogens were added to in vitro mixed

ruminal microorganism fermentations of cows fed

high forage diets, the inclusion of 2% Premidex re-

duced (P < 0.05) E. coli O157:H7 and reduced (P <

0.06) Salmonella Typhimurium populations com-

pared to controls (Figure 2). However, when cows

were fed high grain diets (data not shown) there was

no effect of feedstuff addition on pathogen popula-

tions in ruminal fluid fermentations. In in vitro mixed

fecal microorganism fermentations of cattle fed high

grain diets (Figure 3), E. coli O157:H7 populations

were numerically reduced in hyperimmunized whole

egg and beer well yeast (spray dried) and was sig-

nificantly reduced (P < 0.05) in fermentations con-

taining the CitriStim:PremiDex blend. Salmonella

Typhimurium populations in these in vitro fermenta-

tions were also reduced (P < 0.05) by addition of the

CitriStim:PremiDex blend.

When these by-product feedstuffs were included

in in vitro pig fecal fermentations, PremiDex and the

CitriStim:PremiDex blend reduced (P < 0.05) E. coli

O157:H7 populations more than 10-fold compared

to corn meal controls (Figure 4). However, the ad-

dition of the commercial oligosaccharide product

and hyperimmunized whole egg increased (P < 0.05)

populations of Salmonella Typhimurium more than 1

log10 CFU/ml compared to corn meal controls.

144 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

dISCuSSIon

In recent years, food safety has become an in-

creasingly important issue to animal producers (Da-

vies, 2011; Sargeant, et al., 2007) and to the national

economy as a whole (Scharff, 2010). Research has

shown that diet composition and feeding methods

can affect intestinal microbial populations of cattle,

including populations of foodborne pathogenic bac-

teria, such as E. coli O157:H7 and Salmonella (Bu-

chko, et al., 2000; Diez-Gonzalez, et al., 1998; Fox,

et al., 2007; Jacob, et al., 2008b). Distillers grains

(DG) have seen a rapid increase in inclusion in ani-

mal diets (Klopfenstein, et al., 2008; Richman, 2007),

following the increase in the production of ethanol

from corn fermentation (Richman, 2007). Distill-

ers grains as well as other by-products have some

broad impacts on some members of the microbial

ecosystem (Callaway, et al., 2010; Fron, et al., 1996;

Williams, et al., 2010). Recently, the inclusion of DG

in cattle diets was shown to increase fecal shedding

of E. coli O157:H7 (Jacob, et al., 2008b; Jacob, et

al., 2008c; Wells, et al., 2009). In in vitro studies,

it was found that ruminal fluid from cattle fed DG

supported a higher level of E. coli O157:H7 growth,

and that the inclusion of DG in fermentations also

increased E. coli O157:H7 populations (Jacob, et al.,

2008a). However, results were variable based largely

Figure 3. Effects of feedstuffs on populations of E. coli O157:H7 (light bars) and Salmonella Ty-phimurium when inoculated into mixed fecal microorganism fermentations from cattle fed a grain-based diet. Feedstuffs were included at 2% w/v, and bars represent the mean of 3 fermentations, and error bars indicate standard deviation. Bars marked with superscript a indicate differences from control of P < 0.05.

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 145

upon source of DG (Jacob, et al., 2009; Jacob, et al.,

2010; Wells, et al., 2009). Other researchers found

that inclusion of high levels of corn or wheat DDGS

in feedlot diets of cattle may allow E. coli O157:H7

improved survival in feces (Yang, et al., 2010).

Emerging microbial diversity data indicates that

different gastrointestinal microbial populations are

selected for by different feedstuffs, possibly due to

the presence (or absence) of some limiting compo-

nent in the diet, such as is found in prebiotic feed-

stuffs (Patra and Saxena, 2009; Tajima, et al., 2001).

In the present study, a variety of novel by-product

feedstuffs and prebiotic compounds were examined

to determine their impact on foodborne pathogenic

bacterial populations in pure and mixed cultures of

bacteria from cattle and swine. In general, the novel

by-product feedstuffs did not affect populations of

E. coli O157:H7 or Salmonella in pure or mixed cul-

ture fermentations. In pure cultures, the direct ef-

fects of the different by-products were negligible,

with no feedstuff causing as much as a 10-fold de-

crease in pathogen populations, though a blend of

CitriStim:PremiDex (a prebiotic source of oligosac-

charides such as maltooligosaccharides) did cause a

significant reduction in E. coli O157:H7 populations.

When the prebiotic PremiDex was included in

ruminal fluid fermentations from a cow fed a high

forage diet, populations of both E. coli O157:H7 and

Figure 4. Effects of feedstuffs on populations of E. coli O157:H7 (light bars) and Salmonella Ty-phimurium when inoculated into mixed fecal microorganism fermentations from growing swine fed a commercial finishing diet. Feedstuffs were included at 2% w/v, and bars represent the mean of 3 fermentations, and error bars indicate standard deviation. Bars marked with superscript a indicate differences from control of P < 0.05.

146 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

Salmonella populations were decreased significant-

ly; but this effect disappeared in ruminal fluid from

cattle fed a high grain diet. However, when fecal bac-

teria from cattle fed high grain diets were fermented

with by-products feeds, the CitriStim:PremiDex pre-

biotic blend reduced E. coli O157:H7 populations.

Collectively, these results indicate that CitriStim and/

or PremiDex reduced E. coli O157:H7 populations

and could be an effective dietary additive to reduce

foodborne pathogenic bacteria prior to harvest in

ruminant animals, although further research is need-

ed to clarify the magnitude of the effect in further in

vitro studies and in live animals during feeding trials.

In swine fecal fermentations, prebiotic ad-

dition had a greater impact than in the cattle

ruminal or fecal fermentations. PremiDex and the

CitriStim:PremiDex blend significantly decreased E.

coli O157:H7 populations in swine fecal fermenta-

tions, while no novel by-product feedstuff affected

E. coli O157:H7 populations. However, residual bio-

mass and hyperimmunized whole egg increased S.

Typhimurium populations more than 20-fold in these

in vitro fecal fermentations. The difference in effects

of these feedstuffs between the two pathogenic spe-

cies is likely due to indirect effects on other members

of the microbial population who either outcompete

the pathogen (possibly mediated via a prebiotic-

type effect), or alternatively creation of a competi-

tive “vacuum” in the fecal microbial ecosystem that

is consequently exploited by Salmonella. Further

research is needed to understand the mechanism

behind this impact on pathogen populations.

ConCluSIonS

Collectively, it appears that PremiDex, and the

CitriStim:PremiDex blend had the greatest impact

on foodborne pathogenic bacterial populations in

mixed gastrointestinal microbial fermentations in the

feedstuffs examined presently. Populations of E. coli

O157:H7 in fecal fermentations from cows fed high

grain diets and swine fecal fermentations were most

strongly affected by the CitriStim:PremiDex blend

inclusion. The anti-foodborne pathogen effect ap-

pears to be indirectly mediated by prebiotic effects

on the mixed gastrointestinal microbial consortium

rather than by direct effects against the pathogens.

Further research is needed to clarify the mode of ac-

tion of these and other by-product feedstuffs and

how much impact they have on the mixed microbial

ecosystem when fed to live animals.

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the following fields:

150 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

With an open access publication model of this

journal, all interested readers around the world can

freely access articles online. AFAB publishes origi-

nal papers including, but not limited to the types

of manuscripts described in the following sections.

Papers that have been, or are scheduled to be, pub-

lished elsewhere should not be submitted and will

not be reviewed. Opinions or views expressed in pa-

pers published by AFAB are those of the author(s)

and do not necessarily represent the opinion of the

AFAB or the editorial board.

MAnuSCRIPT TYPeS

Full-Length Research Manuscripts

AFAB accepts full-length research articles con-

taining four (4) figures and/or tables or more. AFAB

emphasizes the importance of sound scientific ex-

perimentation on any of the topics listed in the focus

areas followed by clear concise writing that describes

the research in its entirety. The results of experi-

ments published in AFAB must be replicated, with

appropriate statistical assessment of experimental

variation and assignment of significant difference.

Major headings to include are: Abstract, Introduc-tion, Materials and Methods, Results, Discussion (or Results and Discussion), Conclusion, Acknowl-edgements (optional), Appendix for abbreviations (optional), and References.

Manuscripts clearly lacking in language will be re-

turned to author without review, with a suggestion

that English editing be sought before the paper is

reconsidered. AFAB offers a fee based language

service upon request. Please contact [email protected] for more information about our fees

and services.

Rapid Communications

Under normal circumstances, AFAB aims for re-

ceipt-to-decision times of approximately one month or less. Accepted papers will have priority for publi-

cation in the next available issue of AFAB. However,

if an author chooses or requires a much more rapid

peer review, the journal editorial office has the capa-

bility to shorten the review timing to one week or less.

Any type of manuscript whether it be a full length

manuscript, brief communication or review paper can

be submitted as a rapid communication. There will be

additional costs for processing and page charges will

be double the normal rate. Authors who choose this

option must select Rapid Communications as the pa-

per type when submitting the paper and the editors

will judge whether a rapid review is possible and let

the author know immediately.

Brief Communications

Brief communications are short research notes giv-

ing the results of complete experiments but are con-

sidered less comprehensive than full-length articles

with three (3) figures and/or tables or less. Manuscripts

should be prepared with the same subheadings as full

length research papers. The running head above the

title of the paper is “Brief Communications.”

Unsolicited Review Papers

Review papers are welcome on any topic listed in

the focus section and have no page limits. Reviews

are assessed the same pages charges as all other

manuscripts. All AFAB guidelines for style and form

apply. Major headings to include are: Abstract, In-troduction, Main discussion topics and appropri-ate subheadings, Conclusions, Acknowledgements (optional) and References. Review papers shorter

than 20 pages of double spaced text and references

will be considered mini-reviews with the subhead-

ing above the title on the first page. The running

head above the title of the paper is either “Review”

or “Mini-review”.

Solicited Review Papers

Solicited reviews will have no page limits. The

editor-in-chief will send invitations to the authors

and then review these contributions when they are

submitted. Nominations or suggestions for potential

timely reviews are welcomed by the editors or edito-

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 151

rial board members and should be sent to submit@

afabjournal.com. There will be no page charges for

solicited review papers but the solicitation must origi-

nate from the editor-in-chief or one of the editors. Re-

quests from authors will automatically be classified as

unsolicited review papers. The running head above

the title of the paper will be “Invited Review.”

Conference and Special Issues Reviews

AFAB welcomes opportunities to publish papers

from symposia, scientific conference, and/or meet-

ings in their entirety. Conference organizers need

simply to contact AFAB at [email protected]

and a rapid decision is guaranteed. If in agreement,

the conference organizers must guarantee delivery

of a set number of peer reviewed manuscripts within

a specified time and submitted in the same format

as that described for unsolicited review papers. Con-

ference papers must be prepared in accordance with

the guidelines for review articles and are subject to

peer review. The conference chair must decide

whether or not they wish to serve as Special Issue

Editor and conduct the editorial review process. If

the conference chair/organizer chooses to serve as

special issue editor, this will involve review of the pa-

pers and, if necessary, returning them to the authors

for revision. The conference organizer then submits

the revised manuscripts to the journal editorial of-

fice for further editorial examination. Final revisions

by the author and recommendations for acceptance

or rejection by the chair must be completed by a

mutually agreed upon date between the editor and

the conference organizer. Manuscripts not meeting

this deadline will not be included in the published

symposium proceedings but if submitted later can

still be considered as unsolicited review papers. Al-

though offprints and costs of pages are the same

as for all other papers, the symposium chair may be

asked to guarantee an agreed upon number of hard

copies to be purchased by conference attendees. If

the decision is not to publish the symposium as a

special issue, the individual authors retain the right

to submit their papers for consideration for the jour-

nal as ordinary unsolicited review manuscripts.

Book Reviews

AFAB publishes reviews of books considered to

be of interest to the readers. The editor-in-chief ordi-

narily solicits reviews. Book reviews shall be prepared

in accordance to the style and form requirements of

the journal, and they are subject to editorial revision.

No page charges will be assessed solicited reviews

while unsolicited book reviews will be assigned the

regular page charge rate.

Opinions and Current Viewpoints

The purpose of this section will be to discuss, cri-

tique, or expand on scientific points made in articles

recently published in AFAB. Drafts must be received

within 6 months of an article’s publication. Opinions

and current perspectives do not have page limits.

They shall have a title followed by the body of the

text and references. Author name(s) and affiliation(s)

shall be placed between the end of the text and list

of references. If this document pertains to a par-

ticular manuscript then the author(s) of the original

paper(s) will be provided a copy of the letter and of-

fered the opportunity to submit for consideration a

reply within 30 days. Responses will have the same

page restrictions and format as the original opinion

and current viewpoint, and the titles shall end with

“Opinions.” They will be published together. Letters

and replies shall follow appropriate AFAB format

and may be edited by the editor-in-chief and a tech-

nical editor. If multiple letters on the same topic are

received, a representative set of opinions concern-

ing a specific article will be published. A disclaimer

will be added by the editorial staff that the opinion

expressed in this viewpoint is the authors alone and

does not necessarily represent the opinion of AFAB

or the editorial board.

CoPYRIghT AgReeMenT

The copyright form is published in AFAB as space

permits and is available online (www.afabjournal.com).

AFAB grants to the author the right of re-publication

152 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

in any book of which he or she is the author or edi-

tor, subject only to giving proper credit to the original

journal publication of the article by AFAB. AFAB re-

tains the copyright to all materials accepted for pub-

lication in the journal. If an author desires to reprint

a table or figure published from a non-AFAB source,

written evidence of copyright permission from an au-

thority representing that source must be obtained by

the author and forwarded to the AFAB editorial office.

PeeR ReVIew PRoCeSS

Authors will be requested to provide the names

and complete addresses including emails of five (5) potential reviewers who have expertise in the research

area and no conflict of interest with any of the authors.

Except for manuscripts designated as Rapid Commu-

nication each reviewer has two (2) weeks to review

the manuscript, and submit comments electronically

to the editorial office. Authors have three (3) weeks

to complete the revision, which shall be returned to

the editorial office within six (6) weeks after which the

authors risk having their manuscript removed from

AFAB files if they fail to ask the editorial office for

an extension by email. Deleted manuscripts will be

reconsidered, but they will have to be processed as

new manuscripts with an additional processing fee as-

sessed upon submission. Once reviewed, the author

will be notified of the outcome and advised accord-

ingly. Editors handle all initial correspondence with

authors during the review process. The editor-in chief

will notify the author of the final decision to accept or

reject. Rejected manuscripts can be resubmitted only

with an invitation from the editor or editor-in chief. Re-

vised versions of previously rejected manuscripts are

treated as new submissions.

PRoduCTIon of PRoofS

Accepted manuscripts are forwarded to the edito-

rial office for technical editing and layout. The manu-

script is then formatted, figures are reproduced, and

author proofs are prepared as PDFs. Author proofs

of all manuscripts will be provided to the correspond-

ing author. Author proofs should be read carefully and

checked against the typed manuscript, because the

responsibility for proofreading is with the author(s).

Corrections must be returned by e-mail. Changes

sent by e-mail to the technical editor must indicate

page, column, and line numbers for each correction

to be made on the proof. Corrections can also be

marked using “track changes” in Microsoft Word or

using e-annotation tools for electronic proof correc-

tion in Adobe Acrobat to indicate necessary chang-

es. Author alterations to proofs exceeding 5% of the

original proof content will be charged to the author. All

correspondence of proofs must be agreed to by the

editorial office and the author within 48 hours or proof

will be published as is and AFAB will assume no re-

sponsibility for errors that result in the final publication.

PuBlICATIon ChARgeS

AFAB has two publication charge options: conven-

tional page charges and rapid communication. The

current charge for conventional publication is $25 per printed page in the journal. There is no additional

charge for the publication of pages containing color

images, micrographs or pictures. For authors who

wish to have their papers processed as a rapid com-

munication, authors will pay the rapid communication

fee when proofs are returned to the editorial office

in addition to twice the conventional page charges.

Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one

week and $100 per journal page.

hARd CoPY offPRInTS

If you are wishing to obtain a physical hard copy of

the AFAB journal, offprints are available in any quan-

tity at an additional charge: $100/page for black-white

and $150/page for color prints. You may order your

offprints at any time after publication on our website.

Scientific conference organizers may be expected to

agree to a set number of offprints as a part of their

agreement with AFAB.

MAnuSCRIPT ConTenT ReQuIReMenTS

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 153

Preparing the Manuscript File

Manuscripts must be written in grammatically

correct English. AFAB offers a fee based language

service upon request ([email protected]).

Manuscripts should be typed double-spaced, with

lines and pages numbered consecutively. All docu-

ments must be submitted in Microsoft Word (.doc or

.docx, PC or Mac). All special characters (e.g., Greek,

math, symbols) should be inserted using the sym-

bols palette available in this font. Tables and figures

should be placed in separate sections at the end of

the manuscript (not placed in the text). Failure to fol-

low these instructions will cause delays of the pro-

cessing and review of the manuscript.

Title Page

At the very top of the title page, include a title of

not more than 100 characters. Format the title with

the first letter of each word capitalized. No abbre-

viations should be used. Under the title, the authors

names are listed. Use the author’s initials for both first

and middle names with a period (full-stop) between

initials (e.g., W. A. Afab). Underneath the authors, a

list affiliations must be listed. Please use numerical

superscripts after the author’s names to designate

affiliation. If an authors address has changed since

the research was completed, this new information

must be designated as “Current address:”. The cor-

responding author should be indicated with an aster-

isk e.g., * Corresponding author. The title page shall

include the name and full address of the correspond-

ing author. Telephone and e-mail address must also

be provided for the corresponding author, and email-addresses must be provided for all authors.

Editing

Author-derived abbreviations should be defined

at first use in the abstract and again in the body of

the manuscript. If abbreviations are extensive au-

thors may need to provide a list of abbreviations

at the beginning of the manuscript. In vivo, in vitro

and bacterial names must be italicized (obligatory).

Authors must avoid single sentence paragraphs and

merge such paragraphs appropriately. Authors must

not begin sentences with “Figure or Table shows…”

as these are inanimate objects and cannot “show”

anything. When number are reported in text or in ta-

bles, always put a zero in front of decimal numbers:

“0.10” instead of “.10”.

MAnuSCRIPT SeCTIonS

Abstract

The abstract provides an abridged version of the

manuscript. Please submit your abstract on a sepa-

rate page after the title page. The abstract should

provide a justification of your work, objectives, meth-

ods, results, discussion and implications of study or

review findings . Your abstract must consist of com-

plete sentences without references to other work or

footnotes and must not exceed 250 words. On the

same page as your abstract, please provide at least ten (10) keywords to be used for linking and index-

ing. Ideally, these keywords should include signifi-

cant words from the title.

Introduction

The introduction should clearly present the foun-

dation of the manuscript topic and what makes the

research or the review unique. The introduction

should validate why this topic is important based on

previously published literature, and the relevance of

the current research. Overall goals and project ob-

jectives must be clearly stated in the final sentence

of the last paragraphs of the introduction.

Materials and Methods

Information on equipment and chemicals used

must include the full company name, city, and state

(country if outside the United States or Province if

in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].

Variability, Replication, and Statistical Analysis

To properly assess biological systems indepen-

154 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

dent replication of experiments and quantification

of variation among replicates is required by AFAB.

Reviewers and/or editors may request additional

statistical analysis depending on the nature of the

data and it will be the responsibility of the authors

to respond appropriately. Statistical methods com-

monly used in the bacteriology do not need to be

described in detail, but an adequate description

and/or appropriate references should be provided.

The statistical model and experimental unit must

be designated when appropriate. The experimen-

tal unit is the smallest unit to which an individual

treatment is imposed. For bacterial growth stud-

ies, the average of replicate tubes per single study

per treatment is the experimental unit; therefore,

individual studies must be replicated. Repeated

time analyses of the same sample usually do not

constitute independent experimental units. Mea-

surements on the same experimental unit over time

are also not independent and must not be consid-

ered as independent experimental units. For analy-

sis of time effects, assess as a rate of change over

time. Standard deviation refers to the variability

in the biological response being measured and is

presented as standard deviation or standard error

according to the definitions described in statistical

references or textbooks.

Results

Results represent the presentation of data in

words and all data should be described in same

fashion. No discussion of literature is included in

the results section.

DiscussionThe discussion section involves comparing the

current data outcomes with previously published

work in this area without repeating the text in the

results section. Critical and in-depth dialogue is

encouraged.

Results and Discussion

Results and discussion can be under combined or

separate headings.

Conclusions

State conclusions (not a summary) briefly in one

paragraph.

Acknowledgments

Acknowledgments of individuals should include

institution, city, and state; city and country if not U.S.;

and City or Province if in Canada. Copies being re-

viewed shall have authors’ institutions omitted to re-

tain anonymity.

References

a) Citing References In Text

Authors of cited papers in the text are to be pre-

sented as follows: Adams and Harry (1992) or Smith

and Jones (1990, 1992). If more than two authors of

one article, the first author’s name is followed by the

abbreviation et al. in italics. If the sentence structure

requires that the authors’ names be included in pa-

rentheses, the proper format is (Adams and Harry,

1982; Harry, 1988a,b; Harry et al., 1993). Citations to a

group of references should be listed first alphabeti-

cally then chronologically. Work that has not been

submitted or accepted for publication shall be listed

in the text as: “G.C. Jay (institution, city, and state,

personal communication).” The author’s own un-

published work should be listed in the text as “(J.

Adams, unpublished data).” Personal communica-

tions and unsubmitted unpublished data must not

be included in the References section. Two or more

publications by the same authors in the same year

must be made distinct with lowercase letters after

the year (2010a,b). Likewise when multiple author ci-

tations designated by et al. in the text have the same

first author, then even if the other authors are differ-

ent these references in the text and the references

section must be identified by a letter. For example

“(James et al., 2010a,b)” in text, refers to “James,

Smith, and Elliot. 2010a” and “James, West, and Ad-

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012 155

ams. 2010b” in the reference section.

b) Citing References In Reference Section

In the References section, references are listed in

alphabetical order by authors’ last names, and then

chronologically. List only those references cited in the

text. Manuscripts submitted for publication, accepted

for publication or in press can be given in the refer-

ence section followed by the designation: “(submit-

ted)”, “(accepted)’, or “(In Press), respectively. If the

DOI number of unpublished references is available,

you must give the number. The year of publication fol-

lows the authors’ names. All authors’ names must be

included in the citation in the Reference section. Jour-

nals must be abbreviated. First and last page num-

bers must be provided. Sample references are given

below. Consult recent issues of AFAB for examples

not included in the following section.

Journal manuscript:

Examples:

Chase, G., and L. Erlandsen. 1976. Evidence for a

complex life cycle and endospore formation in the

attached, filamentous, segmented bacterium from

murine ileum. J. Bacteriol. 127:572-583.

Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van

Doesburg, and A. J. M. Stams. 2009. A typical

one-carbon metabolism of an acetogenic and

hydrogenogenic Moorella thermioacetica strain.

Arch. Microbiol. 191:123-131.

Book:

Examples:

Hungate, R. E. 1966. The rumen and its microbes.

Academic Press, Inc., New York, NY. 533 p.

Book Chapter:

Examples:

O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.

Assessing consumer concerns and perceptions

of food safety risks and practices: Methodologies

and outcomes. In: S. C. Ricke and F. T. Jones. Eds.

Perspectives on Food Safety Issues of Food Animal

Derived Foods. Univ. Arkansas Press, Fayetteville,

AR. p 273-288.

dissertation and thesis:

Maciorowski, K. G. 2000. Rapid detection of Salmo-

nella spp. and indicators of fecal contamination

in animal feed. Ph.D. Diss. Texas A&M University,

College Station, TX.

Donalson, L. M. 2005. The in vivo and in vitro effect

of a fructooligosacharide prebiotic combined with

alfalfa molt diets on egg production and Salmo-

nella in laying hens. M.S. thesis. Texas A&M Uni-

versity, College Station, TX.

Van Loo, E. 2009. Consumer perception of ready-to-

eat deli foods and organic meat. M.S. thesis. Uni-

versity of Arkansas, Fayetteville, AR. 202 p.

web sites, patents:

Examples:

Davis, C. 2010. Salmonella. Medicinenet.com.

http://www.medicinenet.com/salmonella /article.

htm. Accessed July, 2010.

Afab, F. 2010, Development of a novel process. U.S.

Patent #_____

Author(s). Year. Article title. Journal title [abbreviated].

Volume number:inclusive pages.

Author(s) [or editor(s)]. Year. Title. Edition or volume (if

relevant). Publisher name, Place of publication. Number

of pages.

Author(s) of the chapter. Year. Title of the chapter. In:

author(s) or editor(s). Title of the book. Edition or vol-

ume, if relevant. Publisher name, Place of publication.

Inclusive pages of chapter.

Author. Date of degree. Title. Type of publication, such

as Ph.D. Diss or M.S. thesis. Institution, Place of institu-

tion. Total number of pages.

156 Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 2, Issue 2 - 2012

Abstracts and Symposia Proceedings:

Fischer, J. R. 2007. Building a prosperous future in

which agriculture uses and produces energy effi-

ciently and effectively. NABC report 19, Agricultural

Biofuels: Tech., Sustainability, and Profitability. p.27

Musgrove, M. T., and M. E. Berrang. 2008. Presence

of aerobic microorganisms, Enterobacteriaceae and

Salmonella in the shell egg processing environment.

IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10)

Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Op-

tions and risks by using diagnostic gene chips. Pro-

gram and abstracts book , The 8th Biennieal Con-

gress of the Anaerobe Society of the Americas. p.

86 (Abstr.)

Data Presentation in Tables and Figures

Figures and tables to be published in AFAB must

be constructed in such a fashion that they are able

to “stand alone” in the published manuscript. This

means that the reader should be able to look at

the figure or table independently of the rest of the

manuscript and be able to comprehend the experi-

mental approach sufficiently to interpret the data.

Consequently, all statistical analyses should be very

carefully presented along with variation estimates

and what constitutes an independent replication

and the number of replicates used to calculate the

averages presented in the table or figure.

Each table and figure must be on a separate

page in the submitted paper. In addition, you will

need to submit all data for charts, tables and

figures in native format when possible (e.g., Mi-

crosoft excel, Powerpoint). Photographs should

be submitted as high-resolution (600 dpi) .jpg or

tif. files. All figures should be clearly presented with

well defined axis and units of measurement. Sym-

bols, lines, and bars must be made distinct as “stand

alone” black and white presentations. Stippling,

dashed lines etc. are encouraged for multiple com-

parison but shades of gray are discouraged. Color

images, micrographs, pictures are recommended

and there is no additional fee for their submission.

AFAB Online Edition is Now Available!

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