Extremophilic exopolysaccharides A review and new

Contents lists available at ScienceDirect

Carbohydrate Polymers

journal homepage: www.elsevier.com/locate/carbpol

Extremophilic exopolysaccharides: A review and new perspectives onengineering strategies and applications

Jia Wanga,d, David R. Salema,b,c,⁎, Rajesh K. Sania,c,d,⁎

a Department of Chemical and Biological Engineering, South Dakota School of Mines and Technology, Rapid City, SD 57701, USAbDepartment of Materials and Metallurgical Engineering, South Dakota School of Mines and Technology, Rapid City, SD 57701, USAc Composite and Nanocomposite Advanced Manufacturing – Biomaterials Center (CNAM-Bio Center), Rapid City, SD 57701, USAd BuG ReMeDEE consortium, South Dakota School of Mines and Technology, Rapid City, SD 57701, USA

A R T I C L E I N F O

Keywords:ExtremophileExopolysaccharideExopolysaccharide propertyExopolysaccharide biosynthesisExopolysaccharide application

A B S T R A C T

Numerous microorganisms inhabiting harsh niches produce exopolysaccharides as a significant strategy tosurvive in extreme conditions. The exopolysaccharides synthesized by extremophiles possess distinctive char-acteristics due to the varied harsh environments which stimulate the microorganisms to produce these biopo-lymers. Despite many bioprocesses have been designed to yield exopolysaccharides, the production of exopo-lysaccharides by extremophiles is inefficient compared with mesophilic and neutrophilic exopolysaccharideproducers. Meanwhile, the industrial development of novel extremophilic exopolysaccharides remains con-strained due to the lack of exploration. In this review, we summarize the structure and properties of variousexopolysaccharides produced by extremophiles, and also discuss potential metabolic and genetic engineeringstrategies for enhanced yield and modified structure of extremophilic exopolysaccharides. Special focus is givento the applications of extremophilic exopolysaccharides in the areas of biomedicine, food industry, and bio-materials via nano-techniques, casting and electrospinning.

1. Introduction

In the past few decades, extremophilic microorganisms and some oftheir metabolites were reported in light of their particular biosyntheticmechanisms, functions, and properties which can permit the strains tobe habitant in extreme niches. Among all the products from ex-tremophiles, exopolysaccharides (EPSs) have led to significant interestdue to the increasing demand for natural polymers in various industrialfields. EPSs are high molecular weight carbohydrate biopolymers,composed of sugar residues, and are secreted by microorganisms intothe surrounding environment, providing certain properties and func-tions useful to the microorganisms (Nicolaus, Kambourova, & Oner,2010; Poli, Anzelmo, & Nicolaus, 2010). The EPS molecular chains havea broad range of molecular weights, and different microorganisms cansynthesize a wide variety of EPSs with a diverse range of functions, suchas intercellular signal transduction, molecular recognition, protectionagainst predation, adhesion, biofilm formation, construction of a com-fortable extracellular environment, and pathogenic processes (Morielloet al., 2003; Nicolaus et al., 1999). Some of the EPSs with valuablephysicochemical properties have already been utilized in industry. Forinstance, among all the reported EPSs, xanthan gum has been most

studied during the past several decades and applied in a variety of in-dustrial areas. In addition to xanthan gum, dextran and gellan gum arecurrently being used in the food industry (Donot, Fontana, Baccou, &Schorr-Galindo, 2012; Rehm, 2010). Bacterial polysaccharides possess agreat diversity of properties that may not be found in more traditionalpolymers of plant origin. Several EPSs have also demonstrated them-selves as useful materials without the environmental disadvantagesassociated with synthetic polymers (Chawla, Bajaj, Survase, & Singhal,2009; Freitas, Alves, & Reis, 2011; Guezennec, 2002).

Currently, it is widely accepted that extremophilic microorganismswill provide a valuable resource for exploitation in novel biotechnolo-gical processes, including synthesis of unique EPSs (Bhalla, Bansal,Kumar, Bischoff, & Sani, 2013; Nicolaus et al., 2010). The environmentsthat extremophiles inhabit are obviously more inhospitable than theenvironmental pressures inducing common mesophilic and neutrophilicmicrobes to secrete their EPSs. Extremophiles have to adapt to hostileenvironments through unique mechanisms, and the biosynthesis ofEPSs is one of their vital survival mechanisms. Extremophilic micro-organisms inhabiting different extreme environments have been re-cognized as promising producers of EPSs, and the examination of EPSproduction by extremophiles (thermophiles, halophiles, alkaliphiles,

https://doi.org/10.1016/j.carbpol.2018.10.011Received 2 August 2018; Received in revised form 20 September 2018; Accepted 4 October 2018

⁎ Corresponding authors at: Department of Chemical and Biological Engineering, South Dakota School of Mines and Technology, Rapid City, SD 57701, USA.E-mail addresses: [email protected] (D.R. Salem), [email protected] (R.K. Sani).

Carbohydrate Polymers 205 (2019) 8–26

Available online 09 October 20180144-8617/ © 2018 Elsevier Ltd. All rights reserved.

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psychrophiles, and acidophiles) has revealed an abundance of novelproperties that may have strong potential in industrial applications(Fig. 1).

Although more and more novel extremophiles have been isolated,and their unique EPSs characterized, the research depth of ex-tremophilic EPSs is still not comparable with EPSs from mesophilic orneutrophilic microorganisms with regard to biosynthetic pathways,regulatory mechanisms, and engineering strategies. It is necessary tomake a comprehensive summarization concerning the structures andcharacteristics of the recently described extremophilic EPSs, which canprovide crucial fundamentals for further exploitation of engineeringstrategies to obtain tailor-made extremophilic EPSs with desired yieldand functions. The targeted cultivation of extremophilic bacteriathrough metabolic and genetic engineering will eventually pave theway for industrial level applications of extremophilic EPSs.

This article reviews the EPSs produced by various kinds of ex-tremophilic bacteria, including an inventory of extremophilic EPSs ofindustrial interest, as well as promising engineering strategies forhigher yield or modified molecular structure of extremophilic EPSs.Moreover, the recent advances in the actual and potential applicationsof EPSs produced by extremophilic bacteria are presented.

2. EPSs produced by different extremophile types

2.1. EPSs produced by thermophiles

Elevated temperature generally increases the rate of most chemicalreactions and improves cumulative production in a given time frame.Thus, thermophiles can be of commercial value in the synthesis ofimportant compounds, and are of growing interest to many sectors ofindustry. Although EPS production is lower than most of the meso-philes, the uncommonly short fermentation process, which is usually nomore than 24 h, makes thermophiles important contenders as com-mercially competitive EPS producers (Kambourova et al., 2009;Radchenkova et al., 2013; Yildiz et al., 2014). The thermophilic strainscan also typically minimize environmental contamination from meso-philic microbial growth, reduce operational maintenance cost, andimprove the efficiency of substrate utilization.

Marine hot springs, terrestrial hot springs, and deep sea thermalvents have been demonstrated as the primary habitats that promotethermophilic microbial organisms, and the majority of EPSs producedby thermophiles have been located in these types of environments.Several thermophilic bacteria in hot marine shallow vents or marine hotsprings have been shown to produce large amounts of EPSs (Mancaet al., 1996; Moriello et al., 2003; Nicolaus et al., 2002; Nicolaus,Moriello, Lama, Poli, & Gambacorta, 2004). These environments aretypically characterized by their high temperature, high pressure, andtoxic, high inorganic or metal concentrations. Thermophilic

microorganisms can survive in high temperatures, and their EPS pro-duction has been a proposed adaptation mechanism to enable theirsurvival in these extreme conditions.

The growth media for thermophiles, containing sugars as carbonand energy sources, have always been considered a primary target to beoptimized for maximum production of EPSs. Disaccharides such asmaltose, lactose and sucrose are the optimized carbon source for mostof thermophilic bacteria for EPS production. Besides chemical compo-sition and molecular weight, thermophilic EPSs have been character-ized mostly in terms of thermostability. The highest decompositiontemperature of 280 °C is from an EPS produced by Geobacillus tepida-mans (Table 1). The summarized data suggest that the type of sugarsubunits present in the EPS may affect their thermostability. Themodification of monomer sugars or some other residues in EPSs can beutilized to find out the active sites for certain functions (e.g., thermo-stability) of EPSs. Although a relatively unexplored area with a sparsedatabase, there is already significant evidence that EPSs from thermo-philes possess a broad range of interesting properties for industrialapplications (Nicolaus et al., 2004, 2010). The literature to date in-dicates that further screening and systematic investigation of EPSsproduced by thermophiles, in conjunction with advances in under-standing the biochemistry of microbial EPS synthesis, will result in thediscovery of novel biopolymers of commercial importance.

2.2. EPSs produced by psychrophiles

Psychrophiles can be isolated from Antarctic, Arctic, or deep-seasediment, and they predominate in marine ecosystems (Ewert &Deming, 2013; Li, Zhou, Zhang, Wang, & Zhu, 2008; Nevot, Deroncele,Montes, & Mercade, 2008; Nichols, Bowman, & Guezennec, 2005). TheEPSs from psychrophilic marine bacteria are generally carboxylatedpolysaccharides, and the carboxyl groups confer a net negative chargeand acidic properties to the EPSs at the pH of seawater (pH around 8)(Caruso et al., 2017; Casillo, Parrilli et al., 2017). The negative chargeof psychrophilic EPSs can also be attributed to the phosphate groups(Corsaro et al., 2004; Llamas et al., 2010). In the marine environment,bacterial EPSs are essential in the production of aggregates, adhesion tosurface, biofilm formation and sequestering of nutrients, and provideprotection and ecosystem stability. Due to their polyanionic property,psychrophilic EPSs can accumulate cations such as metal ions, andmetal binding offers a potential ecological role for these biopolymers.Extracellular polysaccharides strengthen the psychrophiles’ ability tocompete and survive in changing environmental conditions by alteringthe physical and biogeochemical micro-environment around the cells(Nichols, Bowman et al., 2005). The EPSs of psychrophiles in a coldmarine environment should possess the capability to protect the mi-croorganisms from not only the low temperature but also the relativelyhigh salinity (Caruso et al., 2018). Therefore, the EPSs secreted by the

Fig. 1. The EPS from different kinds of extremophiles and potential applications.

J. Wang et al. Carbohydrate Polymers 205 (2019) 8–26

9

Table1

EPSs

from

extrem

ophilic

bacteria.

Extrem

ophiles

(spe

cificco

nditions)

Suga

rcarbon

source

Mon

osacch

arideco

mpo

sition

andlin

kage

pattern

Molecular

weigh

tProp

erties

andactivities

Referen

ce

Thermop

hiles

Geoba

cillu

sthermod

enitri-fi

cans

ArzA-6

(65°C,p

H7.0)

Fruc

tose

•Mon

osacch

aridean

alysis:Man

nose/g

alactose/arabino

se/

fruc

tose/g

luco

se(1/0

.13/

0.1/

0.06

/0.05,

byrelative

ratio)

500kD

aNot

tested

Pano

syan

,DiDon

ato,

Poli,

and

Nicolau

s,(201

8)Geoba

cillu

stoebiiArzA-8

(65°C,

pH7.0)

Fruc

tose

•Mon

osacch

aridean

alysis:Man

nose/g

alactose/g

luco

se/

Arabino

se(1/0

.5/0

.2/0

.05,

byrelative

ratio)

600kD

aNot

tested

Pano

syan

,DiDon

ato,

Poli,

and

Nicolau

s,(201

8)Rho

dothermus

marinus

DSM

4252

T

(65°C,p

H7.2)

Lactose

•Mon

osacch

aridean

alysis:Gluco

se/arabino

se/xylose(1/1

.57/

3.72

,byrelative

ratio)

73.5

kDa

Not

tested

Sardariet

al.(20

17)

Rho

dothermus

marinus

MAT4

93(65°C,p

H7.2)

Maltose

•Mon

osacch

aridean

alysis:Gluco

se/arabino

se/xylose/man

nose

(1/3

.75/

3.02

/1.87,

byrelative

ratio)

85.5

kDa

Not

tested

Sardariet

al.(20

17)

Geoba

cillu

ssp.T

S3-9

(55°C,p

H8.0)

Lactose

•Mon

osacch

aridean

alysis:M

anno

se/g

luco

se/rha

mno

se(1/0

.14/

0.06

,byrelative

prop

ortion

)32

00kD

aAntioxida

ntactivity

Antitum

oractivity

Wan

get

al.(20

17)

Aeribacillus

pallidu

s41

8(55°C,p

H7.0)

Maltose

EPS1

EPS1

700kD

aEP

S2

Abo

ve10

00kD

a

Deg

rada

tion

tempe

rature

EPS1

176°C,E

PS222

6°C

Pseu

doplasticrheo

logicalprop

erty

Foam

ingab

ility

Emulsifyingactivity

Rad

chen

kova

etal.(20

13),

Rad

chen

kova

etal.(20

14),

Rad

chen

kova

etal.(20

15)

•Mon

osacch

aridean

alysis:Man

nose/treha

lose/g

alactosamine/

gluc

osam

ine/ga

lactose/gluc

ose/ribo

se(69.3/

7.8/

6.3/

5.4/

4.7/

3.4/

2.9,

bymolar

ratio)

EPS2

•Mon

osacch

aridean

alysis:Man

nose/g

alactose/g

luco

se/

galactosam

ine/gluc

osam

ine/ribo

se/arabino

se(33.9/

17.9/1

5.5/

11.7/8

.1/5

.3/4

.9,by

molar

ratio)

Brevibacillus

thermorub

er42

3(55°C,p

H6.5)

Maltose

•Mon

osacch

aridean

alysis:Gluco

se/g

alactose/m

anno

se/

galactosam

ine/man

nosamine(57.7/

16.3/9

.2/1

4.2/

2.4,

bype

rcen

tage

ofab

unda

nce)

Not

tested

Bioc

ompa

tibility

Yild

izet

al.(20

14)

Ano

xyba

cillu

ssp.R

4-33

(55°C,p

H8.0)

Gluco

se•M

onosacch

aridean

alysis:M

anno

se/g

luco

se(1/0

.45,

byrelative

prop

ortion

)Abo

ve10

00kD

aBiosorptionof

heav

ymetals

Zhao

etal.(20

14)

Aeribacillus

pallidu

sYM-1

(55°C,

pH7.5)

Gluco

se•M

onosacch

aridean

alysis:Gluco

se/altrose/m

anno

se/g

alactose

(36.6/

30.9/2

4.4/

8.1,

bymolar

ratiope

rcen

tage

)54

0kD

aEm

ulsifyingactivity

Zhen

get

al.(20

12)

Thermus

aqua

ticus

YT-1(60°C,p

H7.5)

Not

adde

d•M

onosacch

aridean

alysis:Galactose/N

-acetylgalactosamine(1/

1,by

molar

ratio)

•Sacch

ariderepe

atingun

it:T

etrasaccha

ride

unit

500kD

aIm

mun

oreg

ulatoryactivity

Linet

al.(20

11)

Geoba

cillu

sthermod

enitri-fi

cans

B3-

72(65°C,p

H7.0)

Sucrose

EPS1

EPS2

400kD

aDeg

rada

tion

tempe

rature

240°C

Hinde

rHSV

-2replicationin

human

periph

eral

bloo

dmon

onuc

lear

cells

andpa

rtially

restorethe

immun

olog

ical

disordersde

term

ined

byHSV

-2

Arena

etal.(20

09),

Nicolau

set

al.(20

00)

•Mon

osacch

aridean

alysis:Gluco

se/m

anno

se(1/0

.3,b

yrelative

ratio)

EPS2

•Mon

osacch

aridean

alysis:Man

nose/g

luco

se(1/0

.2,b

yrelative

ratio)

Geoba

cillu

stepida

man

sV26

4(60°C,p

H7.0)

Maltose

•Mon

osacch

aridean

alysis:Gluco

se/g

alactose/fuc

ose/fruc

tose

(1/0

.07/

0.04

/0.02,

bymolar

ratio)

Abo

ve10

00kD

aDeg

rada

tion

tempe

rature

280°C

Anti-cytotoxicity

Kam

bourov

aet

al.(20

09)

Geoba

cillu

ssp.4

004(60°C,p

H7.0)

Sucrose

EPS1

EPS3

1000

kDa

Not

tested

Moriello

etal.(20

03)

•Mon

osacch

aridean

alysis:G

luco

se/m

anno

se/g

alactose

(1.0/0

.5/

0.3,

byrelative

ratio)

EPS2

•Mon

osacch

aridean

alysis:M

anno

se/g

luco

se/g

alactose

(1.0/0

.3/

trace,

byrelative

ratio)

EPS3

•Mon

osacch

aridean

alysis:Galactose/m

anno

se/g

luco

samine/

arab

inose(1.0/0

.8/0

.4/0

.2,by

relative

ratio)

•Sacch

ariderepe

atingun

it:P

entasaccha

ride

unit

Bacillu

stherman

tarcti-cus(65°C,

pH6.0)

Man

nose

EPS1

EPS2

300kD

aNot

tested

Man

caet

al.(19

96)

•Mon

osacch

aridean

alysis:M

anno

se/g

luco

se(1.0/0

.7,b

yrelative

molar

prop

ortion

)EP

S2

•Mon

osacch

aridean

alysis:Man

nose

•Con

figu

ration

:α-m

anno

(con

tinuedon

next

page)


10

Table1(con

tinued)

Extrem

ophiles

(spe

cificco

nditions)

Suga

rcarbon

source

Mon

osacch

arideco

mpo

sition

andlin

kage

pattern

Molecular

weigh

tProp

erties

andactivities

Referen

ce

Psyc

hrop

hiles

Pseudo

alteromo-na

ssp.M

ER14

4(4

°C,p

H7.0)

Sucrose

•Mon

osacch

aridean

alysis:Gluco

se/m

anno

se/g

luco

samine/

arab

inose/gluc

uron

icacid/g

alacturonicacid/g

alactose

(1/0

.36/

0.26

/0.06/

0.06

/0.05/

0.03

,by

relative

molar

ratio)

250kD

aHeavy

metal

chelation

Cryop

rotectiveactivity

Carusoet

al.(20

18)

Lactobacillus

sakeiT

MW

1.41

1(10°C,p

H5.6)

Sucrose

•Mon

osacch

aridean

alysis:Gluco

se3×

105kD

aNot

tested

Prechtlet

al.(20

18)

Winogradsky

ella

sp.C

AL3

84(4

°C,

pH7.0)

Gluco

se•M

onosacch

aridean

alysis:Gluco

se/m

anno

se/g

alacturonicacid/

arab

inose/ga

lactose/gluc

osam

ine/gluc

uron

icacid

(1/0

.5/0

.3/

0.25

/0.1/0

.1/0

.1,by

relative

prop

ortion

)

Not

tested

Emulsifyingactivity

Cryop

rotectiveactivity

Heavy

metal

chelation

Carusoet

al.(20

17)

Winogradsky

ella

sp.C

AL3

96(4

°C,

pH7.0)

Sucrose

•Mon

osacch

aridean

alysis:Man

nose/arabino

se/g

alacturonic

acid/g

lucu

ronicacid/g

alactose/g

luco

se/g

luco

samine(1/0

.9/

0.4/

0.3/

0.2/

0.2/

0.01

,by

relative

prop

ortion

)

Not

tested

Cryop

rotectiveactivity

Heavy

metal

chelation

Carusoet

al.(20

17)

Colwellia

sp.G

W18

5(15°C,p

H6.0)

Sucrose

•Mon

osacch

aridean

alysis:Gluco

se/m

anno

se/g

alactose/

galactosam

ine/gluc

uron

icacid/g

alacturonicacid

(1/1

/0.7/0

.7/

0.3/

0.04

,byrelative

prop

ortion

)

Not

tested

Cryop

rotectiveactivity

Heavy

metal

chelation

Carusoet

al.(20

17)

Shew

anella

sp.C

AL6

06(4

°C,p

H7.0)

Sucrose

•Mon

osacch

aridean

alysis:Gluco

se/g

alactose/m

anno

se/

galactosam

ine/gluc

uron

icacid/g

alacturonicacid

(1/1

/0.9/0

.6/

0.3/

0.1,

byrelative

prop

ortion

)

Not

tested

Cryop

rotectiveactivity

Heavy

metal

chelation

Carusoet

al.(20

17)

Colwellia

psychrerythraea34

H(4

°C,p

H7.6)

Not

adde

d•T

herepe

atingun

it:T

risaccha

ride

structurewithaN-acetyl-

quinov

osam

inean

dtw

oga

lacturon

icacid

residu

esNot

tested

Inhibitory

effecton

icerecrystallization

Cryop

rotectiveactivity

forthestrain

itself

Casillo,

Parrilliet

al.(20

17),

Marxet

al.(20

09)

Pseudo

alteromo-na

sulvaeTC

14(20°C,p

H7.6)

Not

adde

dEP

S1

EPS1

1000

kDa

EPS2

4000

kDa

Anti-biofi

lmactivity

Brian-Jaissonet

al.(20

16)

•Mon

osacch

aridean

alysis:Gluco

seEP

S2

•Mon

osacch

aridean

alysis:Gluco

sePseudo

alteromo-na

selya

koviiA

rcpo

15(15°C,p

H7.2)

Gluco

se•M

onosacch

aridean

alysis:Man

nose/g

alacturonicacid

(3.3/1

.0,

byrelative

molar

ratio)

17,000

kDa

Cryop

rotectiveactivity

Kim

,Kim

,Park,

andYim

,201

6)

Pseudo

mon

assp.ID1(11°C,p

H7.0)

Gluco

se•M

onosacch

aridean

alysis:Gluco

se/g

alactose/fuc

ose(50.38

/25

.34/

24.28,

byweigh

tpe

rcen

tage

)Abo

ve20

00kD

aEm

ulsifyingactivity

Cryop

rotectiveactivity

forthestrain

itselfas

wellas

forothe

rba

cteria

Pseu

doplasticrheo

logicalprop

erty

Carrión

etal.(20

15)

Cobetia

marinaDSM

Z47

41(20°C,

pH7.6)

Gluco

se•M

onosacch

aridean

alysis:Ribose/3-de

oxy-D-m

anno

-oct-2-

uloson

icacid

(1/1

,bymolar

ratio)

270kD

aNot

tested

Lelcha

tet

al.(20

15)

Polariba

cter

sp.S

M11

27(15°C,p

H7.0)

Gluco

se•M

onosacch

aridean

alysis:N-acetylgluco

samine/man

nose/

Glucu

ronicacid/g

alactose/fuc

ose/gluc

ose/rham

nose

(28.0/

23.4/2

1.4/

17.3/7

.4/1

.6/0

.8,by

molar

percen

tage

)

220kD

aAntioxida

ntactivity

Moisture-retentionab

ility

Pseu

doplasticrheo

logicalprop

erty

Low-tem

perature

protective

effecton

human

derm

alfibrob

lasts

Non

toxican

dno

nirritatingto

skin

Sunet

al.(20

15)

Pseudo

alteromo-na

ssp.S

M20

310

(15°C,p

H7.5)

Gluco

se•M

onosacch

aridean

alysis:Man

nose/g

luco

se/g

alactose/N

-acetylgluc

osam

ine/rham

nose/N

-acetylgalactosamine/xy

lose

(71.7/

10.7/9

.0/4

.0/2

.1/1

.5/0

.9,by

molar

percen

tage

)

Abo

ve20

00kD

aEn

hanc

ethehigh

-salinitytoleranc

eforthestrain

itself

Cryop

rotectionforthestrain

itselfan

dothe

rba

cteria

Liuet

al.(20

13)

Pseudo

alteromo-na

ssp.S

-5(8

°C,

pH7.6)

Gluco

se•M

onosacch

aridean

alysis:Gluco

se/g

alactose/m

anno

se(50.9/

44.3/4

.8,b

ymolar

ratio)

397kD

aIm

mun

oreg

ulatoryactivity

Baiet

al.(20

12)

Pseudo

alteromo-na

ssp.S

M99

13(15°C,p

H7.5)

Lactose

•Mon

osacch

aridean

alysis:6

-Gluco

se,terminal

arab

inofuran

osyl,

term

inal

gluc

opyran

osyl,terminal

galactose,

4-xy

lose,4

-gluco

sean

d3,6-ga

lactose(61.8/

11.0/1

1.2/

3.1/

3.9/

5.0/

4.0,

byweigh

tpe

rcen

tage

)

•The

linka

gebe

tweentherepe

atingsuga

run

its:

α-1,6lin

kage

,an

dthis

EPSwas

structurally

characterizedas

alin

ear

arrang

emen

tofα

-(1,6)-gluco

sean

dahigh

degree

ofacetylation

•The

repe

atingun

it:-6)-[3,6-O-acetyl]-α-D

-Glcp-(1-6)-[3-O

-acetyl]-α-D-G

lcp-(1-6)-[3-O

-acetyl]-α-D

-Glcp-(1-6)-[3-O

-acetyl]-α-D-G

lcp-(1-

40kD

aFu

nction

stab

ilizing

andthermostabilityen

hanc

emen

ton

theproteasessecreted

bythesamestrain

Metal-binding

prop

erty

Floc

culation

prop

erty

Qin,Z

hu,C

hen,

Wan

g,an

dZh

ang,

2007

)

(con

tinuedon

next

page)


11

Table1(con

tinued)

Extrem

ophiles

(spe

cificco

nditions)

Suga

rcarbon

source

Mon

osacch

arideco

mpo

sition

andlin

kage

pattern

Molecular

weigh

tProp

erties

andactivities

Referen

ce

Flavobacterium

frigidarium

CAM00

5(20°C,p

H7.0)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/m

anno

se/g

alactose/

gluc

ose/gluc

uron

icacid/N

-acetyl-g

luco

samine(5/7

4/3/

8/8/

1,w/w

atpe

rcen

tage

oftotalsuga

rs)

1810

kDa

Cryop

rotectan

tformicroorga

nism

sNicho

ls,L

ardièreet

al.(20

05)

Myroidesod

oratus

CAM03

0(20°C,

pH7.0)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/rha

mno

se/xylose/

man

nose/g

alactose/g

luco

se/g

alacturonicacid/g

lucu

ronicacid/

N-acetylgalactosamine/N-acetylgluco

samine(6/1

/2/4

8/4/

9/2/

10/1

0/8,

w/w

atpe

rcen

tage

oftotalsuga

rs)

190kD

aCryop

rotectan

tformicroorga

nism

sNicho

ls,L

ardièreet

al.(20

05)

Polariba

cter

irgensiiCAM00

6(20°C,p

H7.0)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/fuc

ose/man

nose/

galactose/gluc

ose/gluc

uron

icacid/N

-acetylgalactosamine/N-

acetyl-gluco

samine(2/1

1/33

/38/

4/6/

1/4,

w/w

atpe

rcen

tage

oftotalsuga

rs)

2100

kDa

Cryop

rotectan

tformicroorga

nism

sNicho

ls,L

ardièreet

al.(20

05)

Pseudo

alteromo-na

ssp.C

AM00

3(20°C,p

H7.0)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/ribose/rham

nose/fuc

ose/

man

nose/g

luco

se/g

lucu

ronicacid/N

-acetylgalactosamine/N-

acetylgluc

osam

ine(4/2

/6/2

9/40

/16/

1/1/

1,w/w

atpe

rcen

tage

oftotalsuga

rs)

1800

kDa

Cryop

rotectan

tformicroorga

nism

sNicho

ls,L

ardièreet

al.(20

05)

Pseudo

alteromo-na

ssp.C

AM01

5(20°C,p

H7.0)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/rha

mno

se/xylose/

man

nose/g

alactose/g

luco

se/g

lucu

ronicacid/N

-acetyl-

galactosam

ine(10/

6/1/

36/4

/38/

3/3,

w/w

atpe

rcen

tage

oftotalsuga

rs)

2800

kDa

Cryop

rotectan

tformicroorga

nism

sNicho

ls,L

ardièreet

al.(20

05)

Pseudo

alteromo-na

ssp.C

AM02

3(20°C,p

H7.0)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/m

anno

se/g

alactose/

gluc

ose/ga

lacturon

icacid/g

lucu

ronicacid/N

-acetyl-

galactosam

ine/N-acetyl-g

alactosamine(12/

2/1/

75/5

/3/2

,w/w

atpe

rcen

tage

oftotalsuga

rs)

1800

kDa

Cryop

rotectan

tformicroorga

nism

sNicho

ls,L

ardièreet

al.(20

05)

Pseudo

alteromo-na

ssp.C

AM02

5(20°C,p

H7.0)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/ribose/rham

nose/fuc

ose/

man

nose/g

alactose/g

luco

se/g

alacturonicacid/N

-acetyl-

galactosam

ine(3/1

/5/1

/1/5

/52/

30/1

,w/w

atpe

rcen

tage

oftotalsuga

rs)

5700

kDa

Cryop

rotectan

tformicroorga

nism

sNicho

ls,L

ardièreet

al.(20

05)

Pseudo

alteromo-na

ssp.C

AM03

6(20°C,p

H7.0)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/m

anno

se/g

alactose/

gluc

ose/ga

lacturon

icacid/N

-acetyl-g

alactosamine/N-acetyl-

gluc

osam

ine(3/2

4/1/

26/3

0/14

/1,w/w

atpe

rcen

tage

oftotal

suga

rs)

1700

kDa

Cryop

rotectan

tformicroorga

nism

sNicho

ls,L

ardièreet

al.(20

05)

Pseudo

alteromo-na

ssp.C

AM06

4(20°C,p

H7.0)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/m

anno

se/g

alactose/

gluc

ose/gluc

uron

icacid/N

-acetyl-g

alactosamine/N-acetyl-

gluc

osam

ine(4/6

4/4/

8/6/

11/2

,w/w

atpe

rcen

tage

oftotal

suga

rs)

100kD

aCryop

rotectan

tformicroorga

nism

sNicho

ls,L

ardièreet

al.(20

05)

Shew

anella

livingstonensisCAM09

0(20°C,p

H7.0)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/rha

mno

se/xylose/

man

nose/g

alactose/g

luco

se/g

lucu

ronicacid/N

-acetylga

lactosam

ine(13/

2/2/

41/5

/10/

20/7

,w/w

atpe

rcen

tage

oftotalsuga

rs)

80kD

aCryop

rotectan

tformicroorga

nism

sNicho

ls,L

ardièreet

al.(20

05)

Pseudo

alteromo-na

sha

loplan

ktis

TAC12

5(15°C,p

H7.5)

Not

adde

d•M

onosacch

aridean

alysis:M

anno

se/g

luco

se(1/trace,b

yrelative

ratio)

Not

tested

Not

tested

Corsaro

etal.(20

04)

Pseudo

mon

assp.N

CMB20

21(17°C,p

H7.5)

Gluco

seEP

S1

Not

tested

Metal

cation

precipitation

Christensen

,Kjosbak

ken,

andSm

idsrød

,19

85)

•Mon

osacch

aridean

alysis:Gluco

se/g

alactose/g

lucu

ronicacid/

galacturon

icacid

(1/0

.81/

0.42

/0.32,

bymolar

ratio)

EPS2

•Mon

osacch

aridean

alysis:N-acetylgluco

samine/2-ke

to-3-

deox

yoctuloson

icacid/u

nide

ntified

6-de

oxyh

exose(1/1

/1,by

molar

ratio)

Halothe

rmop

hiles

(con

tinuedon

next

page)


12

Table1(con

tinued)

Extrem

ophiles

(spe

cificco

nditions)

Suga

rcarbon

source

Mon

osacch

arideco

mpo

sition

andlin

kage

pattern

Molecular

weigh

tProp

erties

andactivities

Referen

ce

Halom

onas

nitroreducensWB1

(60°C,p

H7.5,

5%w/v

NaC

l)Gluco

seEP

S1

EPS152

00kD

aEP

S2

30kD

aEP

S3

1.3kD

a

Emulsifyingactivity

Antioxida

ntactivity

Heavy

metal

bind

ingcapa

city

Pseu

doplasticrheo

logicalprop

erty

Chikk

anna

,Gho

sh,a

ndKisho

re,(201

8)

•Mon

osacch

aridean

alysis:Gluco

se/m

anno

se/g

alactose/xylose

(28/

64/6

/traces,

byweigh

tpe

rcen

tage

)EP

S2

•Mon

osacch

aridean

alysis:Gluco

se/m

anno

se/rha

mno

se/

arab

inose/xy

lose

(18.5/

44/2

/1.5/traces,by

weigh

tpe

rcen

tage

)EP

S3

•Mon

osacch

aridean

alysis:Gluco

se/m

anno

se/g

alactose/

galacturon

icacid/fructose(19/

56.5/1

4.2/

1.5/

traces,b

yweigh

tpe

rcen

tage

)Ba

cillu

slicheniform

isB3

-15(45°C,

pH7.0,

2%w/v

NaC

l)Gluco

seEP

S1

EPS2

600kD

aAntiviral

andim

mun

oreg

ulatoryactivities

Span

òan

dArena

(201

6),A

rena

etal.

(200

6),

Mau

geri

etal.(20

02)

•Mon

osacch

aridean

alysis:Man

nose/g

luco

se(1.0/0

.3,by

molar

ratio)

EPS2

•Mon

osacch

aridean

alysis:Man

nose

•Rep

eating

unit:T

etrasaccha

ride

EPS3

•Mon

osacch

aridean

alysis:Gluco

seBa

cillu

slicheniform

isT1

4(50°C,

pH8.0,

5%w/v

NaC

l)Su

crose

•Mon

osacch

aridean

alysis:Fruc

tose/fuc

ose/gluc

ose/

galactosam

ine/man

nose

(1.0/0

.75/

0.28

/trace/trace,b

yrelative

molar

ratio)

•Sacch

ariderepe

atingun

it:T

risaccha

ride

unit

•Ano

meric

confi

guration

:β-m

anno

-pyran

osidic

confi

guration

1000

kDa

Deg

rada

tion

tempe

rature

240°C

Anti-cytotoxicity

Visco

elasticity

Antiviral

andim

mun

omod

ulatoryeff

ects

against

herpes

simplex

virustype

2(H

SV-2)

Anti-biofi

lmactivity

Emulsifyingan

dstab

ilizing

activities

Span

ò,La

ganà

,Visalli,

Mau

geri,a

ndGug

liand

olo,

(201

6),

Gug

liand

oloet

al.(20

13),

Span

òet

al.(20

13)

Geoba

cillu

ssp.1

A60

(50°C,p

H8.0,

5%w/v

NaC

l)Su

crose

•Mon

osacch

aridean

alysis:Man

nose/g

alactose/g

alactosamine/

fuco

se/g

luco

se(1/0

.69/

0.65

/0.59/

0.35

,byrelative

prop

ortion

)Not

tested

Heavy

metal

bind

ingcapa

city

Gug

liand

olo,

Lentini,Sp

anò,

and

Mau

geri,2

012)

Halop

hiles

Chrom

ohalobac-te

rcana

densis28

(30°C,p

H8.5,

15%

w/v

NaC

l)

Lactose

•Mon

osacch

aridean

alysis:Gluco

samine/gluc

ose/rham

nose/

xylose/u

nkno

wnsuga

r(36.7/

32.3/2

5.4/

1.7/

3.9,

byweigh

tpe

rcen

tage

)

Abo

ve10

00kD

aDeg

rada

tion

tempe

rature

250°C

Pseu

doplasticrheo

logicalprop

erty

Highsw

ellin

gbe

havior

Emulsifyingan

dstab

ilizing

activities

Foam

ingab

ility

Rad

chen

kova

etal.(20

18)

Halolactib

aci-llusmiurensisSE

ENMKU3(32°C,p

H8.0,

75g/

LNaC

l)

Gluco

se•M

onosacch

aridean

alysis:Galactose/g

luco

se/xylose/fruc

tose/

man

nose/rha

mno

se(61.87

/25.17

/not

tested

/not

tested

/not

tested

/not

tested

,byrelative

percen

tage

)

Not

tested

Antioxida

ntactivity

Arunet

al.(20

17)

Kocuria

roseaZJ

UQH

(30°C,p

H7.0,

5.8%

w/v

MgS

O4)

Not

adde

d•M

onosacch

aridean

alysis:Gluco

se56

.59kD

aNot

tested

Gu,

Jiao

,Wu,

Liu,

andChe

n,20

17)

Vibrioalgino

lyticus

CNCM

I-49

94(25°C,p

H7.2,

30g/

Lsea

salts)

Gluco

se•M

onosacch

aridean

alysis:Galacturonicacid/N

-acetyl-

gluc

osam

ine(3/1

,byrelative

ratio)

1160

kDa

Not

tested

Drouilla

rdet

al.(20

15)

Halom

onas

smyrnensisAAD6T

(37°C,p

H7.0,

137.2g/

LNaC

l)

Sucrose

•Mon

osacch

aridean

alysis:Fruc

tose

Abo

ve10

00kD

aDeg

rada

tion

tempe

rature

253°C

Biofl

occu

lating

activity

Anti-cytotoxicity

Bioc

ompa

tibility

Antitum

oractivity

afterpe

riod

ateox

idation

Sarilm

iser

andOne

r(201

4),

Küç

ükaşik

etal.(20

11),

Sam

etal.(20

11),

Poliet

al.(20

09)

Alteromon

asmacleod

ii(28°C,p

H7.2,

30g/

Lseasalts)

Gluco

se•M

onosacch

aridean

alysis:Galactose/g

luco

se/rha

mno

se/

gluc

uron

icacid/g

alacturonicacid/m

anno

se/fuc

ose(5.9/2

.6/

2.5/

2.0/

1.9/

1.4/

1.0,

bymolar

ratio)

1100

kDa

Not

tested

LeCostaou

ëcet

al.(20

12)

(con

tinuedon

next

page)


13

Table1(con

tinued)

Extrem

ophiles

(spe

cificco

nditions)

Suga

rcarbon

source

Mon

osacch

arideco

mpo

sition

andlin

kage

pattern

Molecular

weigh

tProp

erties

andactivities

Referen

ce

Halom

onas

almeriensisM8T

(32°C,

pH7.0,

7.5%

w/v

totalsalts)

Gluco

seEP

S1

EPS1

6300

kDa

EPS2

15kD

a

Emulsifyingactivity

Heavy

metal

bind

ingcapa

city

Pseu

doplasticrheo

logicalprop

erty

Llam

aset

al.(20

12)

•Mon

osacch

aridean

alysis:Man

nose/g

luco

se/rha

mno

se(72/

27.5/0

.5,b

yweigh

tpe

rcen

tage

)EP

S2

•Mon

osacch

aridean

alysis:Man

nose/g

luco

se(70/

30,by

weigh

tpe

rcen

tage

)Vibriosp.Q

Y10

1(25°C,p

H7.0,

3.0%

w/v

NaC

l)Alginate

•Mon

osacch

aridean

alysis:Rha

mno

se/g

alacturonicacid/

gluc

uron

icacid/g

luco

samine/ga

lactose/gluc

ose/fuco

se/

man

nose

(23.90

/23.05

/21.47

/12.15

/6.89/

6.57

/3.61/

2.36

,by

molar

percen

tage

)

546kD

aBiofi

lmform

ationinhibition

activity

Pre-existing

biofi

lmdisrup

tion

activity

Jian

get

al.(20

11)

Halom

onas

stenophila

B100

(32°C,

pH7.2,

7.5%

w/v

marine

salts)

Gluco

se•M

onosacch

aridean

alysis:Gluco

se/g

alactose/m

anno

se(44.5/

40.5/1

5.0,

byweigh

tpe

rcen

tage

)37

5kD

aAntitum

oractivity

afterov

ersulpha

tion

Ruiz-Ruizet

al.(20

11)

Halom

onas

stenophila

N12

T(32°C,

pH7.2,

7.5%

w/v

marine

salts)

Gluco

se•M

onosacch

aridean

alysis:Gluco

se/fuc

ose/man

nose

(48.82

/25

.69/

25.47,

byweigh

tpe

rcen

tage

)25

0kD

aAntitum

oractivity

afterov

ersulpha

tion

Ruiz-Ruizet

al.(20

11)

Salip

iger

mucosus

A3T

(32°C,p

H7.0,

2.5%

w/v

totalsalts)

Gluco

se•M

onosacch

aridean

alysis:Man

nose/g

alactose/g

luco

se/fuc

ose

(34/

32.9/1

9.7/

13.4,by

weigh

tpe

rcen

tage

)25

0kD

aEm

ulsifyingactivity

Heavy

metal

bind

ingcapa

city

Pseu

doplasticrheo

logicalprop

erty

Llam

aset

al.(20

10)

Idiomarinafontislapido

siF2

3T

(32°C,p

H7.2,

7.5%

w/v

total

salts)

Gluco

seEP

S1

EPS115

00kD

aEP

S2

15kD

a

Emulsifyingactivity

Heavy

metal

bind

ingcapa

city

Pseu

doplasticrheo

logicalprop

erty

Mataet

al.(20

08)

•Mon

osacch

aridean

alysis:Man

nose/g

luco

se/g

alactose/xylose

(46.35

/28.25

/14.85

/trace,by

molar

percen

tage

)EP

S2

•Mon

osacch

aridean

alysis:Man

nose/g

luco

se/g

alactose/xylose

(40/

40/2

0/trace,

bymolar

percen

tage

)IdiomarinaramblicolaR22

T(32°C,

pH7.2,

7.5%

w/v

totalsalts)

Gluco

seEP

S1

EPS1

550kD

aEP

S2

20kD

a

Emulsifyingactivity

Heavy

metal

bind

ingcapa

city

Pseu

doplasticrheo

logicalprop

erty

Mataet

al.(20

08)

•Mon

osacch

aridean

alysis:Man

nose/g

luco

se/rha

mno

se(68.2/

25/6

.8,b

ymolar

percen

tage

)EP

S2

•Mon

osacch

aridean

alysis:M

anno

se/g

alacturonicacid/gluc

ose/

rham

nose/xylose(53.6/

25.29/

18.9/trace/trace,by

molar

percen

tage

)Alteromon

ashispan

icaF3

2T(32°C,

pH7.2,

7.5%

w/v

totalsalts)

Gluco

se•M

onosacch

aridean

alysis:Man

nose/g

luco

se/xylose/rham

nose

(62.75

/18.15

/12.25

/6.85,

bymolar

percen

tage

)19

,000

kDa

Emulsifyingactivity

Heavy

metal

bind

ingcapa

city

Pseu

doplasticrheo

logicalprop

erty

Mataet

al.(20

08)

Halom

onas

euriha

linaF2

-7(32°C,

pH7.2,

7.5%

w/v

totalsalts)

Gluco

se•M

onosacch

aridean

alysis:Gluco

se/m

anno

se/rha

mno

se(2.9/

1.5/

1,by

relative

ratio)

Not

tested

Emulsifyingactivity

Pseu

doplasticrheo

logicalprop

erty

Martíne

z-Che

ca,To

ledo

,ElMab

rouk

i,Que

sada

,an

dCalvo

,(20

07),

Bejar,

Calvo

,Moliz,D

iaz-Martine

z,an

dQue

sada

,(199

6)Halom

onas

ventosae

A11

2T(32°C,

pH7.2,

7.5%

w/v

totalsalts)

Gluco

se•M

onosacch

aridean

alysis:Gluco

se/m

anno

se/g

alactose

(1.75/

4/1,

bymolar

ratio),a

ndsm

allq

uantitiesof

xylose,a

rabino

sean

dga

lacturon

icacid

53kD

aEm

ulsifyingactivity

Heavy

metal

bind

ingcapa

city

Biofi

lmform

ationcapa

city

Pseu

doplasticrheo

logicalprop

erty

Mataet

al.(20

06)

Halom

onas

ventosae

A11

6(32°C,p

H7.2,

7.5%

w/v

total

salts)

Gluco

se•M

onosacch

aridean

alysis:Gluco

se/m

anno

se/g

alactose

(1.25/

4/1,

bymolar

ratio),a

ndsm

allq

uantitiesof

xylose,a

rabino

sean

dga

lacturon

icacid

52kD

aEm

ulsifyingactivity

Heavy

metal

bind

ingcapa

city

Biofi

lmform

ationcapa

city

Pseu

doplasticrheo

logicalprop

erty

Mataet

al.(20

06)

Halom

onas

anticariensis

FP35

T(32°C,p

H7.2,

7.5%

w/

vtotalsalts)

Gluco

se•M

onosacch

aridean

alysis:Gluco

se/m

anno

se/g

alacturonicacid

(1/3

/2.5,b

ymolar

ratio)

20kD

aEm

ulsifyingactivity

Heavy

metal

bind

ingcapa

city

Biofi

lmform

ationcapa

city

Pseu

doplasticrheo

logicalprop

erty

Mataet

al.(20

06)

(con

tinuedon

next

page)


14

Table1(con

tinued)

Extrem

ophiles

(spe

cificco

nditions)

Suga

rcarbon

source

Mon

osacch

arideco

mpo

sition

andlin

kage

pattern

Molecular

weigh

tProp

erties

andactivities

Referen

ce

Halom

onas

anticariensisFP

36(32°C,p

H7.2,

7.5%

w/v

total

salts)

Gluco

se•M

onosacch

aridean

alysis:Gluco

se/m

anno

se/g

alacturonicacid

(1/2

.5/2

.2,by

molar

ratio)

46kD

aEm

ulsifyingactivity

Heavy

metal

bind

ingcapa

city

Biofi

lmform

ationcapa

city

Pseu

doplasticrheo

logicalprop

erty

Mataet

al.(20

06)

Halom

onas

mau

raS-30

(32°C,p

H7.0,

2.5%

w/v

seasalts)

Gluco

se•M

onosacch

aridean

alysis:Man

nose/g

alactose/g

luco

se/

gluc

uron

icacid

(34.8/

14/2

9.3/

21.9,by

weigh

tpe

rcen

tage

)47

00kD

aHeavy

-metal

uptake

Visco

sifyingpo

tential

Pseu

doplasticrheo

logicalprop

erty

Arias

etal.(20

03)

Aph

anothece

haloph

yticaGR02

(30°C,p

H7.0,

1M

NaC

l)Not

adde

dEP

S1

EPS2

Abo

ve20

00kD

a

Gellin

gprop

erty

Strong

affinity

formetal

ions

Liet

al.(20

01)

•Mon

osacch

aridean

alysis:Arabino

se/rha

mno

se/fuc

ose/

man

nose/g

luco

se/g

alactose

(trace/0

.06/

0.05

/0.08/

1/0.75

,by

molar

ratio)

andgluc

uron

icacid

3.58

%of

polysaccha

ride

dry

weigh

tEP

S2

•Mon

osacch

aridean

alysis:Arabino

se/fuc

ose/man

nose/g

luco

se(1/2

.08/

1.57

/2.87,

bymolar

ratio)

andgluc

uron

icacid

15.78%

ofpo

lysaccha

ride

dryweigh

tAlteromon

assp.1

644(25°C,p

H7.0,

30g/

Lseasalts)

Fruc

tose

•Mon

osacch

aridean

alysis:G

alactose/g

luco

se/g

lucu

ronicacid/3

-O-[(R

)-1-carbox

yethyl]-D-glucu

ronicacid/g

alacturonicacid

(1.0/0

.92/

0.7/

0.34

/0.26,

bymolar

ratio)

Not

tested

Gellin

gprop

erty

Samain,

Mile

s,Bo

zzi,Dub

reuc

q,an

dRinau

do,1

997)

Haloa

lkaliphiles

Halom

onas

sp.C

RSS

(30°C,p

H9.0,

100g/

LNaC

l)Acetate

a•M

onosacch

aridean

alysis:Gluco

se/fructose/gluc

osam

ine/

galactosam

ine(1/0

.7/0

.3/trace,by

relative

prop

ortion

)Not

tested

Visco

sity

abov

e0.5η

Solution

viscositycanincrease

atpH

2-3with2.5%

(w/v

)NaC

l

Poliet

al.(20

04)

Bacillu

ssp.(37

°C,p

H10

.5,4

0g/

LNaC

l)Gluco

se•M

onosacch

aridean

alysis:D-galactopy

ranu

ronicacid,2

,4-

diacetam

ido-2,4,6-trideo

xy-D

-gluco

pyrano

se,2-acetam

ido-2-

deox

yD-m

anno

pyranu

ronicacid

andD-galactopy

ranu

ronicacid

withthecarbox

ylgrou

pam

ide-lin

kedto

glycine

•The

repe

atingun

it:-3)-a-d-G

alpA

(Gly)-(1-4)-b-d-Man

pNAcA

-(1-

4)-a-d-G

alpA

-(1-3)-a-d-Q

uipN

Ac4

NAc-(1-

Not

tested

Not

tested

Corsaro,G

rant,G

rant,M

arcian

o,an

dPa

rrilli,(199

9),D

uckw

orth,Grant,

Jone

s,an

dVan

Steenb

erge

n,19

96)

Alkaliphiles

Crono

bacter

saka

zakii(30

°C,p

H10

)Su

crose

•Mon

osacch

aridean

alysis:Gluco

se/m

anno

se/g

alactose/xylose/

arab

inose(14/

24/1

4/20

/1.9,by

weigh

tpe

rcen

tage

)37

60kD

aDeg

rada

tion

tempe

rature

280°C

Pseu

doplasticrheo

logicalprop

erty

Emulsifyingactivity

Jain

etal.(20

12)

Bacillu

scereus

(23°C,p

H10

.5)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/xylose/man

nose/

galactose/gluc

ose/N-acetylgluco

samine(5.0/3

.4/7

0.3/

12.1/

4.7/

4.5,

bymolar

percen

tage

)

Abo

ve16

7kD

aCalcite

bind

ing

Perryet

al.(20

05)

Bacillu

sthuringiensis(23°C,p

H10

.5)

Gluco

se•M

onosacch

aridean

alysis:Arabino

se/rha

mno

se/xylose/

galacturon

icacid/m

anno

se/ga

lactose/gluc

ose(9.4/3

.2/5

.6/

7.5/

52.2/1

6.9/

5.2,

bymolar

percen

tage

)

Abo

ve16

7kD

aCalcite

bind

ing

Perryet

al.(20

05)

aNon

-sug

arcarbon

source.


15

microorganisms in a marine environment may provide both cryopro-tection and a buffering effect against low temperature and high salinitysimultaneously. The secondary molecular structure analysis of psy-chrophilic EPS indicates that a pseudohelicoidal structure may be ad-vantageous for the inhibition of ice recrystallization (Casillo, Parrilliet al., 2017). Moreover, the decoration by amino acid motifs onto themonosaccharide moieties was speculated to endow a structural equili-brium between hydrophilic and hydrophobic regions in the EPS mole-cule, and thus contribute to the inhibitory effect on ice crystal devel-opment (Casillo, Ståhle et al., 2017). The sulphate moieties inpsychrophilic EPSs may also play a significant role against extremelycold environments (Nichols, Guezennec et al., 2005; Nichols, Bowmanet al., 2005). The physical, rheological, and chemical properties of EPSscan be influenced by the length of the polymer chain, and the highmolecular weights of EPSs from psychrophiles provide greater oppor-tunity for complex entanglement of polymer chains and intramolecularassociations, which may contribute to the tertiary structure and en-hance the physical behavior of the EPSs in their environment (Nichols,Bowman et al., 2005). Besides, the EPSs with higher molecular weightalso possess better water binding capacity than EPSs with lower mo-lecular weight (Prechtl, Wefers, Jakob, & Vogel, 2018).

Normally, psychrophilic EPS production can be inhibited by a re-latively elevated temperature, in the region of 20 °C and above (Nichols,Garon, Bowman, Raguenes, & Guezennec, 2004, 2005). The contents ofmonosaccharide components in psychrophilic EPSs can be modifiedthrough change of temperature, and some of the monosaccharides andother residues in EPSs from psychrophiles may help to confer ad-vantageous cryoprotectant properties. For example, the uronic acidcontent in the EPSs produced by Pseudoalteromonas sp. CAM025 at−2 °C and 10 °C was significantly higher than that at 20 °C; and themonosaccharide compositions were also found to differ among the EPSsharvested at −2 °C, 10 °C, and 20 °C (Nichols, Bowman et al., 2005).The psychrotolerant strain Lactobacillus sakei TMW 1.411 produceddextran with less branching and higher molecular weight at 10 °C thanthe dextran produced at 30 °C (Prechtl et al., 2018). At temperaturesbelow the optimum temperature for cell growth, the psychrophiles werestimulated to produce excessive EPSs (Marx, Carpenter, & Deming,2009; Nevot et al., 2008; Nichols et al., 2004). This is consistent withthe fact that EPS production is one of the main mechanisms to protectextremophiles and enable them to survive in extreme conditions.Therefore, output of EPS for each cell can be enhanced with the dete-rioration of environmental conditions in a certain range, albeit the cellgrowth may sharply decrease. Enhancing net EPS production may thusinvolve identifying the optimal trade-off between increased EPS pro-duction per cell and reduced cell count.

In several former studies, the stabilization effect of psychrophilicEPS for protease against thermal denaturation was confirmed (Huston,Methe, & Deming, 2004; Junge, Eicken, Swanson, & Deming, 2006;Marx et al., 2009), which indicates that psychrophilic biopolymers canbe applied to the stabilization of industrially promising enzymes used inunfavorable conditions. In future research on psychrophilic EPSs, it isrecommended that significant insights may be found by comparing thestructure and function of EPSs from different culture conditions, inorder to reveal what kind of structure can be more advantageous forprotection and stabilization effects.

2.3. EPSs produced by halophiles

Moderately halophilic bacteria are defined as those which growoptimally in media containing 5–20% (w/v) salts, and they constitutethe most important eubacteria group living in hypersaline habitats(Ollivier, Caumette, Garcia, & Mah, 1994; C. Qian et al., 2018). Mosthalophilic EPSs are heteropolysaccharides, and mannose and glucoseare the most common monosaccharide moieties in halophilic EPSs(Table 1). So far, the research focus for halophilic EPSs properties hasbeen emulsifying activity, gelling properties, heavy metal binding

capacity, and rheological properties, with existing and potential ap-plications in a range of industrial fields, such as utilization as a sub-stitute for xanthan gum in the food industry.

Changes in salinity affects the biosynthesis of halophilic EPSs,especially the ratio for each type of monosaccharide composition. Toprotect the microorganism from increasing salinity, the content of somemonosaccharide components in EPS may need to be modified in orderto maintain its functions. For the EPS obtained from strain Aphanothecehalophytica GR02, the proportions of galactose and rhamnose decreasedwhen the NaCl concentration in the medium was elevated from 0.5 to2.0 M; in contrast, the proportions of arabinose and glucose increasedwith NaCl concentration. Meanwhile, the monosaccharides present inthe EPS at different salinities stayed the same (P. Li, Liu, & Xu, 2001).This indicates that the increase of glucose and arabinose, and the de-crease of galactose and rhamnose in the EPS secreted by Aphanothecehalophytica GR02 may be advantageous to its survival in a high salinityenvironment. Mata et al. (2006) mentioned that for the strain Halo-monas ventosae A112T, its EPS incorporated a significant quantity ofsulphate. Sulphate is not commonly found in mesophilic EPSs; however,it has been observed in the EPSs excreted by microorganisms living insaline habitats. In addition, the EPSs from halophiles usually containsignificant amounts of uronic acids. The high viscosity of the EPS so-lution at acidic pH and the gelification capacity may be due to the highuronic acid content (Béjar, Llamas, Calvo, & Quesada, 1998). EPSs withhigh concentrations of charged components (e.g. uronic acids) oftenform gels in the presence of metal ions and have enormous potential forremoving toxic metal from polluted environments and wastewater as analternative to other physical and chemical methods.

2.4. EPSs produced by acidophiles

Acidophiles are extremophiles which inhabit a low pH environment,usually less than pH 3 for optimum growth. Some of the acidophilescannot grow at all in a neutral pH condition (Baker-Austin & Dopson,2007; Johnson, 1998; Johnson, Joulian, d’Hugues, & Hallberg, 2008).Both natural and artificial acidic niches can occur in the biosphere, suchas a sulfidic mine area or a marine volcanic vent. The acidic environ-ments usually include the presence of sulphur, sulphide, and theiroxidates. Pyrite is one of the main acidic niches for acidophiles. Theseareas are quite toxic due to high concentrations of various heavy metalsulphides, but they are rich in valuable metals, such as Fe, Cu, Co, Al,Mg, Zn, and Mn (Dopson, Baker-Austin, Koppineedi, & Bond, 2003; Jiaoet al., 2010; Johnson et al., 2008; Nicolaus et al., 2010).

Compared with the research for other kinds of extremophilic EPSs,the acidophilic EPSs have not been studied sufficiently to reveal theirfermentation process, molecular structure, or properties. Usually EPSsfrom acidophiles are considered as bioproducts generated in anotherbioprocessing technology such as a bioleaching process. For acid-ophiles, the genome analysis cannot identify ubiquitous DNA adapta-tions for growth in an extremely low pH environment (Baker-Austin &Dopson, 2007). On the other hand, the EPSs produced by acidophilesmay play a protective role against stress conditions related to the lowpH and presence of metals. Acidophilic EPS biosynthesis can be in-hibited by increased temperature during the bioleaching process, andthe inhibited EPS production may have been related to the loss ofbioleaching efficiency observed in the reactor when the temperaturewas increased (d’Hugues et al., 2008). This phenomenon indicates thatthe acidophilic EPSs protecting acidophiles from an acidic environmentare not able to protect them against a relatively high temperaturecondition, unlike thermophilic EPSs. Therefore, it is of significant in-terest to explore the acidophilic EPSs for functional diversity elucida-tion through molecular level structure and comparison among acid-ophilic and other extremophilic EPSs as models.

Some acidophilic EPSs were discovered during the study of extra-cellular polymeric substances, which are one of the major componentsin biofilms, and they mainly consist of EPSs, proteins, and nucleic acids


16

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(Flemming & Wingender, 2010; Moreno-Paz, Gómez, Arcas, & Parro,2010; Subramanian, Yan, Tyagi, & Surampalli, 2010; Vu, Chen,Crawford, & Ivanova, 2009). The extracellular polymeric substancescontaining acidophilic EPSs are usually generated by mixed culturesduring the bioleaching process. Bioleaching uses the oxidation ability ofbacteria to dissolve metal sulphides in order to facilitate the extractionand recovery of precious metals from primary ores and concentrates.The involved microbial consortia are mainly composed of acidophilic,autotrophic iron-oxidizing, and sulphur-oxidizing bacteria (Michelet al., 2009). In Zeng’s report (Zeng et al., 2010), an acidophilic mixedculture was able to produce extracellular polymeric substances duringthe bioleaching process, and Acidithiobacillus caldus and Leptospirillumferriphilum were considered as the dominant microorganisms in themixed culture. The extracellular polymeric substance had protein,polysaccharide, fatty acid, and ferric ion as its main components.Rhamnose, fucose, xylose, mannose, glucose, and uronic acids were thecomponents of the polysaccharide which could be considered to comefrom the EPS excreted by the mixed culture during the bioleachingprocess. The percentages of these components varied at different sam-pling time during bioleaching, while the presence of these componentsremained stable. A pure culture, Thiobacillus ferrooxidans, was alsocarried in the bioleaching process, and the monosaccharide units of thecarbohydrate in the extracellular polymeric substance were rhamnose,fucose, xylose, mannose, glucose, and glucuronic acid. This composi-tion varied greatly when T. ferrooxidans was grown in a differentmedium containing iron (II) sulphate, pyrite, or sulfur as the solidsubstrate (Gehrke, Telegdi, Thierry, & Sand, 1998).

2.5. EPSs produced by alkaliphiles

The alkaliphiles are microorganisms that grow optimally or verywell at pH values above 9, often between 10 and 12, but cannot grow orgrow slowly at near-neutral pH values (Horikoshi, 1999). Soda lakesand deserts represent the most stable, naturally occurring alkaline en-vironments which can be found all over the world (Rees, Grant, Jones,& Heaphy, 2004). The enzymes isolated from alkaliphiles, includingalkaline proteases, amylases, cellulases, and lipase, have been appliedin various industrial sectors such as the detergent industry (Ito et al.,1998). As with other kinds of extremophiles, the alkaliphiles produceEPSs as metabolic products. So far, certain functions of EPSs from al-kaliphiles have been partially studied (Table 1), but more research onmolecular structure, properties, and the biosynthesis pathway of alka-liphilic EPSs are necessary to improve scientific understanding and toenable targeted industrial applications.

Alkaliphilic EPSs are functional for the attachment of the associatedmicrobial strains to a certain matrix. For example, the binding strengthto calcite was found to be due to the chemical properties of the EPSssecreted by two natural alkaliphiles isolated from biofilms on historiclimestone. Meanwhile, these two alkaliphilic EPSs could also contributeto calcite dissolution in the biofilm development process (Perry et al.,2005). Unlike most other extremophiles, for which sugar is the optimalcarbon source for EPS production, the most efficient carbon source forEPS production of the haloalkalophilic strain Halomonas sp. CRSS wasacetate. The growth conditions strongly influenced the cumulativeproduction, relative fractions of different monosaccharides, andmonomer compositions of the EPS from Halomonas sp. CRSS (Poli et al.,2004).

3. Metabolic and genomic engineering of extremophilic EPSs

Extremophilic EPSs have increasing significance in material andbiomedical applications that require a more profound understanding ofthe metabolic pathways and biosynthetic mechanisms of EPS in order tocontrol the production process and molecular structure, and hence thephysiochemical properties. The development of engineered EPS-pro-ducing strains can also reduce their exceptionally expensive production

costs, allowing extremophilic EPSs to compete in the biopolymermarket. Several biopolymers from mesophiles and neutrophiles, such ascellulose, alginate, gellan, and sphingan have already been profoundlystudied for the enzymes and genes involved in their biosyntheticpathways (Ates, 2015; Schmid, Sieber, & Rehm, 2015). However, theinformation about metabolic pathways and functional assignment ofgene clusters for extremophilic EPS biosynthesis is still limited. Re-search focusing on genetically modified strains capable of producinghighly improved levels of extremophilic EPS is also necessary since,compared to mesophilic and neutrophilic EPS-producing strains, ex-tremophilic bacteria are relatively inefficient at producing EPSs.

3.1. Engineering strategies in EPS biosynthetic pathways for improved EPSproduction and modified molecular weight

EPS biosynthesis is highly associated with catabolic processes ofoxidation and does not interfere with other anabolic bioprocesses(Chawla et al., 2009). As a carbon and energy intensive process, thebiosynthesis of extremophilic EPSs usually requires the recruitment ofnucleoside diphosphate saccharides (NDP-sugars) as precursors, glyco-syltransferases (GTs) for assembly, and membrane proteins for thetransfer of repeat units across cell envelope. Generally, the EPS bio-synthetic pathway starts from glycolysis of simple sugar for cytosolicformation of the NDP-sugar precursors; then the monosaccharides aresequentially transported from nucleotide-sugar donors to activated lipidcarriers and assembled as repeating units of polysaccharide throughGTs. Finally, the EPS needs to be exported to an extracellular en-vironment. Based on the general biosynthetic pathway of EPS, the genesinvolved can be organized into three functional types: (1) genes in-volved in NDP-sugar synthesis, (2) genes coding for GTs required forbiosynthesis of EPS repeating unit, (3) genes encoding proteins forpolymerization and export (Ates, 2015).

During the first phase of EPS biosynthesis, the NDP-sugars representthe interface between primary and secondary metabolism (Ates, Arga,& Oner, 2013). A bottleneck is the low level of activated NDP-sugarprecursors which can be exploited as design space through metabolicengineering to alter the expression of enzymes involved in the centralmetabolism for supplying nucleotide-sugar precursors. The higher EPSproducing mutant demonstrated that the specific activities of phos-phoglucomutase, UDP-glucose pyrophosphorylase, UDP-glucose dehy-drogenase, and UDP-galactose-4-epimerase were higher than those inthe wild-type strain (Fig. 2), indicating these enzymes involved in NDP-sugar synthesis can be potential targets for enhancement of EPS pro-duction (Li et al., 2010; Welman, Maddox, & Archer, 2006; Zhu et al.,2014). Although it is still relatively nascent for extremophilic bacteriato be applied as intact platforms for metabolic engineering, a group ofextremophiles have already been metabolically engineered for en-hanced biofuel or enzyme production due to the recent expansion of thegenetic systems and tools for extremophiles (Lin & Xu, 2013; Zeldeset al., 2015). In EPS-producing bacteria, the sugar substrates are eitherconverted into EPS synthesis or cell mass by alternative intermediarymetabolic routes. The rerouting of the carbon flux through the aug-mentation of a critical enzyme at the principal branch point to NDP-sugar synthesis was considered as a strategy to enhance the EPS pro-duction of several mesophilic bacteria. The homologous over-expres-sion of phosphoglucomutase in Sphingomonas sanxanigenens strain re-sulted in a 17% increase in EPS production (Huang et al., 2013).However, the flow of carbon towards the synthesis of EPS by Sphingo-monas sp. strain S7 was manipulated by augmenting the cellularphosphoglucomutase activity with additional genes, and no significantincrease in EPS yield was observed (Thorne, Mikolajczak, Armentrout,& Pollock, 2000). The over-expression of UDP-glucose pyropho-sphorylase involved in the synthesis of UDP-glucose also had negligibleeffect on EPS productivity. Meanwhile, the inactivation of glucose-6-phosphate dehydrogenase could not divert carbon flow toward EPSsynthesis (Sá-Correia et al., 2002). On the other hand, the simultaneous


17

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over-expression of the UDP-glucose pyrophosphorylase gene andphosphoglucomutase gene was found to promote EPS production inboth Streptococcus thermophilus and Bacillus licheniformis (Levander,Svensson, & Radstrom, 2002; Liu, Chen, Yang, Li, & He, 2017), in-dicating single interventions in metabolic pathways may not be suffi-cient to improve the production of desired EPSs, while multiple inter-ventions are more likely to be efficient. However, with reference toother studies, the increased enzyme specific activities for improvementof UDP-sugar precursor availability is still controversial for guaran-teeing higher EPS production (Boels, Ramos, Kleerebezem, & de Vos,2001; Fialho et al., 2008). Extremophilic EPS production usually ac-companies all the growth phases against the extreme environments,which can be different from the factors inducing mesophilic or neu-trophilic EPS production. It should be cautioned that over-expression ofthe genes specific for NDP-sugar synthesis could generate a metabolicburden for the growth of extremophilic strains.

For the second stage of EPS biosynthesis, glycosyltransferasestransfer the activated nucleotide sugar precursors to the polysaccharidechain on a membrane-associated anchor for elongation. A significantlyincreased EPS production without any deleterious effect to growth wasachieved by over-expression of the gene encoding priming GT forlinking the first galactose moiety to the lipid carrier in a mesophilicstrain Sinorhizobium meliloti (Jones, 2012). The combination of in-creasing EPS precursor availability with engineered GTs in the EPSbiosynthetic route may synergistically enhance the production of ex-tremophilic EPS. In addition, the metabolic control analysis can beperformed for extremophilic EPS biosynthetic machinery to reveal thecontrol points, in order to disclose the most efficient metabolic en-gineering strategy and combine it with highly activated GT system forelevated EPS production (Boels et al., 2001).

After the assembly process, these hydrophilic macromolecules need

to be exported out of the cell membranes to validate their functionssuch as protection against extreme environments. The secretion processrequires a multi-component transport system for the export of carbo-hydrates with complex molecular structures. The intracellular poly-merization and transport of microbial EPSs mainly follow three me-chanisms (Fig. 3): (1) Wzx/Wzy-dependent pathway, (2) ATP-bindingcassette (ABC) transporter-dependent pathway, and (3) synthase-de-pendent pathway (Ates, 2015).

The Wzx/Wzy-dependent pathway is considered as a major me-chanism for Gram-negative bacteria to produce various EPSs. Most ofthe EPSs assembled by the Wzx/Wzy-dependent pathway are hetero-polysaccharides due to the presence of multiple GTs (Schmid et al.,2015). The assembled EPS is translocated across the cytoplasmicmembrane by Wzx flippase and polymerized by Wzy polymerase. Fi-nally, the EPS is transported by polysaccharide co-polymerase (PCP)and outer membrane polysaccharide export (OPX) protein (Islam &Lam, 2014). This transport system was not considered as a major rate-limiting step for EPS biosynthesis, instead the concomitant gene over-expression of the PCP and OPX proteins generated higher molecularweight EPS compared with that of the wild-type strain. Besides, it wasalso disclosed that the higher ratio of over-expressed PCP-OPX proteinsto Wzy polymerase could shift toward the polymerization of longer EPSchains, and vice versa (Galván et al., 2013).

ABC transporter-dependent pathway uses ABC transporter to exportthe EPS across inner membrane instead of Wzx and Wzy proteins, andthe final secretion is still mediated by PCP and OPX proteins as Wzx/Wzy-dependent pathway (Whitney & Howell, 2013). ABC transporter-dependent pathway was considered as mainly presenting in capsularpolysaccharide biosynthesis (Schmid et al., 2015). However, thermo-philic strain Brevibacillus thermoruber 423 was suggested to be followingABC transporter-dependent pathway for its EPS transport due to the

Fig. 2. Potential engineering targets for increasing monosaccharide precursors for EPS biosynthesis in extremophiles. PTS: phosphotransferase system; ABC: ATP-binding cassette transporter; GDP: guanosine diphosphate; UDP: uridine diphosphate; TDP: thymidine diphosphate.


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presence of associated genes in its genome (Yildiz, Radchenkova, Arga,Kambourova, & Oner, 2015). The ABC transporter-dependent pathwayin this Gram-positive thermophilic bacterium diverged from that inwell-studied mesophilic Gram-negative bacteria due to the appearanceof tetratricopeptide repeat (TPR) protein instead of PCP, indicating thatdifferent tactics are required for further engineering of extremophilicstrains compared with mesophilic strains for improvement of EPS se-cretion. The ABC transporter might also be able to dictate the EPS chainlength (Schmid, 2018). Acquisition of high-resolution crystal structuresof these proteins involved in extremophilic EPS polymerization andtransportation will be significant for generating mechanisms and pro-viding information for engineering strategies (Morgan, Strumillo, &Zimmer, 2012).

In the synthase-dependent pathway, the repeating units of EPS arepolymerized and then translocated by a synthase or synthase complex.Due to the reduced enzyme or enzyme system in this process comparedwith other pathways, the molecular structure of EPSs synthesizedthrough the synthase-dependent pathway is simplified, and the syn-thase-dependent pathway is quite favorable for biosynthesis of homo-polysaccharides or simple heteropolysaccharides with only two mono-saccharide units (Schmid et al., 2015). Therefore, it can be speculatedthat the extremophilic EPSs with highly diverse monosaccharide com-position could not be produced through the synthase-dependentpathway. For the mesophilic EPS synthesized through the synthase-dependent mechanism, no correlation was observed between thenumber of synthase complexes and the EPS production level (Maleki,Almaas, Zotchev, Valla, & Ertesvåg, 2016). Meanwhile, the higher ex-pression of the genes encoding EPS synthase complex could link toincreased molecular weight of the EPS (Díaz-Barrera, Soto, &Altamirano, 2012).

The cyclic diguanylic acid (c-di-GMP) is a bacterial secondarymessenger enhancing the activity of EPS synthase with a c-di-GMPbinding domain, and this regulation mechanism is significantly dif-ferent from other types of EPS synthetic pathways (Maleki et al., 2016;Morgan et al., 2012). The upregulation of c-di-GMP level can be astrategy to increase EPS production through synthase-dependentpathway. The mutant with removal of the gene coding for tyrosinephosphatase which repressed the activity of diguanylate cyclase re-sponsible for c-di-GMP synthesis demonstrated 28-fold more EPS pro-duction (Ueda & Wood, 2009). The downregulation of the activity ofphosphodiesterase which degraded c-di-GMP could also be another wayto increase the activity of EPS synthase (Hammer & Bassler, 2009).

The c-di-GMP has been found as an activator for EPS biosynthesis byseveral acidophilic bacteria including Acidithiobacillus species andLeptospirillum ferriphilum (Christel et al., 2018; Díaz, Castro, Copaja, &Guiliani, 2018; Ruiz, Castro, Barriga, Jerez, & Guiliani, 2011). This

phenomenon indicates those acidophilic EPSs might be producedthrough the synthase-dependent pathway. Intriguingly, besides acid-ophilic EPSs, until now none of the other types of extremophilic EPSswere discovered to be using the synthase-dependent pathway for as-sembly and secretion, or utilizing c-di-GMP as a stimulator to promoteEPS production. Therefore, it would be valuable to disclose if those EPSsynthases could be more acidoresistant than the enzymes and trans-porters within Wzx/Wzy- and ABC transporter-dependent pathway, andwhy the EPSs synthesized through the synthase-dependent pathwaycould provide protection against extreme acidic condition.

The biosynthesis of EPSs can also take place extracellularly throughthe dissociated enzymes generated and secreted by the bacterial cells.This EPS synthetic process does not compete with cell growth for ac-tivated monosaccharide precursors or lipid carriers (Prechtl et al.,2018). Halophilic bacterium Halomonas smyrnensis AAD6T was able tosynthesize levan as EPS through a secreted levansucrase. The over-ex-pression of levansucrase could be attained by boric acid as a stimulatorthrough the quorum sensing based signaling effect (Sarilmiser, Ates,Ozdemir, Arga, & Oner, 2015). Moreover, the gene encoding phos-phocarrier protein of the phosphoenolpyruvate sugar phospho-transferase system (PTS) for fructose uptake was knocked out in Halo-monas smyrnensis AAD6T, and the mutant strain displayed an almostthreefold higher efficiency profile of levan production compared withthe wild-type strain (Aydin, Ozer, Oner, & Arga, 2018). The supple-mentation of mannitol to the culture medium also reduced the meta-bolic requirement of fructose in Halomonas smyrnensis AAD6T since themannitol could be directly converted to fructose intracellularly (Ateset al., 2013). Both of these two strategies inhibited the uptake offructose and thus accumulated more fructose moieties extracellularly asprecursors for levan biosynthesis (Fig. 2).

3.2. Monosaccharide component modification strategies

Up to the recent reports, the modification of monosaccharide unitsin the EPS backbone could be attained mainly using three differenttactics, which pave the way for the production of tailor-made ex-tremophilic EPSs. During the first phase within EPS production, thesynthesis of a certain type of NDP-sugar precursor can be weakened tochange the monosaccharide contents in the EPS backbone. A mesophilicbacterium Paenibacillus elgii was identified containing two genes codingfor uridine diphosphate-glucuronic acid (UDP-GlcA) decarboxylase,which could transfer UDP-GlcA to UDP-xylose. The single-geneknockout mutant of UDP-GlcA decarboxylase produced the EPS withhigher glucuronic acid and lower xylose content compared with that ofthe wild-type strain. Meanwhile no significant variation in mannoseand glucose content was observed between the EPSs produced by the

Fig. 3. Potential engineering targets for higherproduction and modified chain length duringEPS assembly and transportation in ex-tremophiles. NDP: nucleoside diphosphate; GT:glycosyltransferase; Wzx: flippase; Wzy: poly-merase; PCP: polysaccharide co-polymerase;OPX: outer membrane polysaccharide exportprotein; ABC: ATP-binding cassette trans-porter; TPR: tetratricopeptide repeat protein; c-di-GMP: cyclic diguanylic acid.


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single-gene knockout mutant and wild-type strain (Li et al., 2015).Another genetic engineering strategy targets the genes coding for

glycosyltransferases responsible for monomer assembly. The condi-tional reduction of the activity of the priming GT involved in attachingthe first monosaccharide unit to the lipid carrier in mesophilic bac-terium Lactobacillus rhamnosus could decrease the amount of repeatingunit modules available for polymerase, leading to premature chaintermination; thus, shorter EPS chains were secreted (Bouazzaoui &LaPointe, 2006). The removal of gene encoding a non-priming GT wasable to block the addition of the undesired monosaccharide unit ontothe EPS polymer chain. In a current study concerning the EPS biosyn-thetic machinery in a mesophilic strain Paenibacillus polymyxa, thestate-of-the-art CRISPR-Cas9 genome editing tool was applied for geneknockout strategy in order to modify its EPS monomer composition.The EPS variants with altered monosaccharide distribution and rheo-logical behavior from the wild-type EPS were obtained by disruption ofthe gene coding for one of the non-priming glycosyltransferases withinits EPS biosynthetic system (Rütering et al., 2017).

The heterologous expression of the exopolysaccharide gene clusterto the recombinant strain also leads to generation of the EPS with dif-ferent monosaccharide composition from the native strain. The genecluster with functional regions coding for EPS biosynthesis regulatoryprotein, glycosyltransferases, EPS chain-length determinator, poly-merase and transporter from a thermotolerant bacterium Streptococcusthermophilus Sfi6 was expressed heterologously in a non-EPS-producingstrain Lactococcus lactis MG1363. The transferred EPS biosyntheticsystem could utilize the NDP-sugars generated in the host strainthrough its house-keeping genes as building blocks for EPS production.For the recombinant EPS, the N-acetylgalactosamine (GalNAc) moietiesin the backbone was replaced by galactose residues, since the hoststrain was unable to synthesize the corresponding UDP-GalNAc pre-cursor. Meanwhile, the recombinant EPS also lacked the galactose side-chain compared with the wild-type EPS (Stingele et al., 1999). Thisheterologous strategy requires that the polymerases and transportersfrom the native strain can recognize the recombinant EPS chain withoutstrong exclusive selectivity. Many extremophilic bacteria are able togenerate two or three EPSs with various molecular weight andmonomer distributions (Table 1). Therefore, the polymerization andexport system from the extremophiles with multi-EPS producing cap-ability may not be strictly specific towards a single type of EPS back-bone, and this infidelity provides a further degree of flexibility inproducing recombinant extremophilic EPS in an engineered strainthrough heterologous genetic engineering techniques.

3.3. Genome annotation for extremophilic EPS biosynthetic system

The genome annotation for extremophilic strains can be a powerfultool to disclose the essential genes associated with EPS biosynthesis andhence gain more insight about the biological mechanisms of EPS pro-duction, serving as the starting point to develop genetic and metabolicengineering strategies to optimize EPS production and modify EPSmonomer composition to fit industrial and biomedical requirements.Currently, a preliminary model of the EPS biosynthesis mechanism wassuccessfully proposed for a thermophilic bacterium Brevibacillus ther-moruber 423 through whole-genome analysis (Yildiz et al., 2015). Thegenome annotation of halophilic strain Halomonas smyrnensis AAD6T

revealed the presence of a Pel exopolysaccharide gene cluster in itsgenome, indicating its capacity to produce Pel EPS besides being alevan producer (Diken et al., 2015). The genome analysis was alsocarried for acidophilic bacterium Leptospirillum ferriphilum, which de-monstrated cellulose and Pel EPS synthetic genes involved in the syn-thase-dependent pathway (Christel et al., 2018). With a relativelylarger genome size, the psychrophile Phormidesmis priestleyi BC1401contained both gene clusters following the scheme of Wzx/Wzy-de-pendent and ABC transporter-dependent EPS export systems (Chrismas,Barker, Anesio, & Sánchez-Baracaldo, 2016). This is quite intriguing,

and further study for EPS characterization and transcriptomics is highlyrecommended to elucidate the EPS biosynthetic and regulatory me-chanism in this psychrophilic bacterium.

4. Recent progress in the application of extremophilic EPSs

4.1. Biomedical application

4.1.1. Antitumor and immunoregulatory effectCurrently, accumulated evidence has demonstrated that ex-

tremophilic EPSs have a broad spectrum of biological activities, such asanti-cancer, anti-oxidant and immunoregulatory properties, which canbe promising for biomedical applications. The anti-cancer efficacy hasalready been recognized for the polysaccharides generated by fungi,algae, and plants (Zong, Cao, & Wang, 2012). Extremophilic EPSs canhardly be highly cytotoxic against malignant proliferating mammaliancells like chemotherapeutic drugs. Instead, extremophilic EPSs mayinduce apoptosis in tumor cells via coupling specific surface receptors(Ruiz-Ruiz et al., 2011). The thermophilic EPSs from Geobacillus sp.TS3-9 was found to significantly inhibit the proliferation of hepatomacarcinoma cell in a dose-dependent manner in vitro (Wang et al., 2017).Further study of antiproliferation effects on non-tumor cells is requiredto identify its antitumor specificity. Furthermore, the addition of non-sugar functional groups onto extremophilic EPS molecules may gen-erate improved biological activities (e.g. antitumor activity) comparedwith native EPSs. An oversulphated EPS produced by a halophilicbacterium Halomonas stenophila B100 could specifically induce apop-tosis of leukemia cells from peripheral blood, and the addition of sul-phate moieties to native EPS was considered to enhance its anti-proliferative efficacy (Ruiz-Ruiz et al., 2011). Another halophilic EPSlevan was modified through periodate oxidation to harbor aldehydegroups, and the aldehyde-activated levan derivatives showed bothbiocompatibility to non-tumor cells and anti-cancer activity againstseveral human tumor cell lines in vitro. The antitumor efficacy wasconfirmed to be enhanced by increasing the oxidation degree of the EPS(Sarilmiser & Oner, 2014).

EPSs can also inhibit tumor progression through the im-munoenhancement effect (Zong et al., 2012). The EPS from a thermo-philic bacterium Thermus aquaticus YT-1 was proved to be an im-munomodulator which stimulated macrophage cells to produce thecytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6).This thermophilic EPS also induced macrophages to release nitric oxide(NO) as inflammatory mediator. Toll-like receptor 2 (TLR2) expressedon the immune cell surface was confirmed as the natural receptor of thisthermophilic EPS (Lin et al., 2011). A psychrophilic EPS produced byPseudoalteromonas sp. strain S-5 showed a similar stimulation effect onmacrophages to secrete TNF-α and NO. Meanwhile, it could also en-hance the phagocyte function of macrophage cells (Bai et al., 2012).The stimulation of the production of TNF-α and NO indicates a role ofthese EPSs in activation of macrophages into the M1 subtype (classi-cally activated macrophages), which can suppress tumor growth, me-tastasis and angiogenesis (Chen et al., 2018). Thus, these extremophilicEPSs have high potential for application in macrophage-mediated im-mune therapy for cancer treatment.

Several extremophilic EPSs were found to be non-cytotoxic fornormal cell lines. The heteropolysaccharide produced by thermophilicbacterium Brevibacillus thermoruber strain 423 demonstrated high bio-compatibility to a monkey kidney fibroblast cell line (Yildiz et al.,2014). Levan secreted by halophile Halomonas smyrnensis AAD6T alsoshowed high biocompatibility and affinity with non-cancerous celllines. The lack of branch structure in this halophilic levan molecule wasconsidered as crucial for the absence of cytotoxic activity (Küçükaşiket al., 2011; Poli et al., 2009). The EPSs from halothermophilic bacteriaBacillus licheniformis strain T14 and B3-15 were non-cytotoxic towardhuman peripheral blood mononuclear cells (PMBC) at maximum con-centration of 400 and 300 μg/ml, respectively (Arena et al., 2006;


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Gugliandolo, Spanò, Lentini, Arena, & Maugeri, 2013). Additionally,thermophilic EPS synthesized by Geobacillus thermodenitrificans strainB3-72 was also non-toxic to PMBC cells at concentration of 300 μg/mlor below (Arena et al., 2009). Psychrophilic EPS of Pseudoalteromonassp. S-5 had no cytotoxic effect under 500 μg/ml against murine peri-toneal macrophages (Bai et al., 2012). These non-cytotoxic ex-tremophilic EPSs are highly promising for application as a biocompa-tible carrier to conjugate antineoplastic drug and targeting ligand suchas monoclonal antibody. This three-phase antitumor drug conjugatewould attain the specific delivery of non-selective cytotoxic drugs totumor tissue with enhanced antitumor efficacy and selectivity(Asamoah-Asare, Zhang, & Chen, 2013; Dragojevic, Ryu, & Raucher,2015). The biodegradability of these extremophilic EPSs must beidentified in order to make in vivo test and future clinical trial feasible.

The immunoregulatory effect of several extremophilic EPSs alsoleads to antiviral activity. The EPSs from halothermophilic strainBacillus licheniformis strain B3-15, Bacillus licheniformis strain T14, andthermophile Geobacillus thermodenitrificans strain B3-72 decreasedherpes simplex virus type 2 (HSV-2) replication in PMBC through sti-mulating the expression of different proinflammatory cytokines in-volved in the immune surveillance toward virus infection, indicating apotential application as therapy in herpes virus infection and im-munocompromised host (Arena et al., 2006, 2009; Spanò & Arena,2016).

4.1.2. Antioxidant effectAntioxidant activity leads to scavenging reactive oxygen species

(ROS), which generate oxidative stress to neuronal cells and are deeplyassociated with chronic and degenerative diseases such as neurode-generative disorders (Xu, Bi, & Wan, 2016). Polysaccharides have beenshown to play a crucial role as natural antioxidants for the preventionof oxidative damage in the human body (Wang et al., 2013). Ex-tremophilic EPSs are usually non-pathogenic and their high biodiversityoffers various biotechnological activities including antioxidancy. Ha-lophilic EPS isolated from Halolactibacillus miurensis, and thermophilicEPS from Geobacillus sp. strain TS3-9 both demonstrated dose-depen-dent scavenging activity against DPPH (2,2-diphenyl-1-picrylhydrazyl),hydroxyl and superoxide free radicals (Arun et al., 2017; Wang et al.,2017). Additionally, the EPS produced by halothermophilic bacteriumHalomonas nitroreducens WB1 had antioxidant properties to scavengehydroxyl and DPPH radicals (Chikkanna, Ghosh, & Kishore, 2018). Ahyper-branched psychrophilic EPS secreted by Polaribacter sp. SM1127showed substantially higher antioxidant activity than that of hyaluronicacid, an industrial annexing agent for scavenging radicals (Sun et al.,2015). These extremophilic EPSs with remarkable antioxidative capa-city may be efficacious in the treatment of neurodegenerative diseases.Furthermore, they can be studied as natural dietary antioxidants for theinactivation of oxyradicals and reduction of the incidence rate of neu-rodegenerative disease.

4.2. Food application

The microbial polysaccharides which are able to stabilize emulsionsbetween water and hydrophobic compounds have potential as naturalemulsifiers in the food industry (Freitas et al., 2009). Bioemulsifiershave the advantages of biodegradability, low toxicity, selectivity andenvironmental compatibility over artificial products (Mata et al., 2008).The emulsifying activity has been found to be common in extremophilicEPSs, including those produced by thermophiles, psychrophiles, halo-philes, and alkaliphiles; and they are all heteropolysaccharides(Table 1). Among those extremophilic EPSs, EPS from psychrophilicbacterium Pseudomonas sp. ID1 had a higher emulsifying activity thanxanthan gum and arabic gum for several food oils (Carrión, Delgado, &Mercade, 2015).

In industrial processes, emulsifiers may be exposed to extremes oftemperature, environmental pH and salinity (Freitas et al., 2009). An

important property of bioemulsifiers from extremophiles is their highemulsion stability over a wide range of temperature, pH and salinity(Zheng et al., 2012). For example, in Arias’ report, neither the viscositynor the pseudoplasticity of mauran (a halophilic EPS) solution was af-fected by the presence of salts, sugar, surfactants, lactic acid, changes inpH, or freezing and thawing (Arias et al., 2003). The minor content ofprotein in those extremophilic EPSs might be essential for the emulsi-fying activity (Llamas, Amjres, Mata, Quesada, & Béjar, 2012). Thepresence of uronic acid and acetyl group in EPS was also considered tocontribute to its emulsifying capacity (Caruso et al., 2017; Jain, Mody,Mishra, & Jha, 2012; Mata et al., 2006).

Pseudoplastic rheological behavior is another common propertyamong those extremophilic EPSs with emulsifying activity (Table 1).High pseudoplasticity is an attractive rheological characteristic in di-verse types of food formulations, such as sauce, dairy, cake, saladdressing, syrup, and pudding (Bahram Parvar & Razavi Seyed, 2012;Han et al., 2014). For the development of foodstuffs, the pseudoplasticproperty of EPS is advantageous to generate comfortable sensoryproperties such as mouth feel and flavor release. It is also useful for foodprocesses, such as mixing, pouring and pumping with different opera-tive shear rates (Han et al., 2014).

Based on current extremophilic EPS research, it remains difficult forthe newly discovered extremophilic EPSs to displace the commercia-lized biopolymers such as xanthan and gellan in the food industry, dueto the costly production processes of extremophilic EPSs using puresugar as substrate. Inexpensive and renewable substrates need to beused for EPS production, such as molasses which can be a feasiblesubstitute for sucrose (Küçükaşik et al., 2011; Sam et al., 2011). Acontinuous cultivation technique with economic potential for industrialscale production also showed a much higher efficiency for the fer-mentation of extremophilic EPS in comparison with batch cultures(Radchenkova et al., 2015).

4.3. Biomaterial application

4.3.1. EPS-based nanoparticlesNanotechnology is an indispensable discipline in the modern

pharmaceutical field for a variety of applications. The non-ideal bio-degradability and biocompatibility of the synthetic nanoparticlesusually impair renal excretion and induce many unacceptable side ef-fects. During the constant lookout for novel products with improvedpharmaceutical functions, the extremophilic EPSs have received in-creasing attention as alternatives to synthetic polymers in the produc-tion and modification of nanoparticles. Their valuable biological char-acteristics and simplicity of chemical modifications make them strongcandidates for nanoparticle application (Raveendran et al., 2014).

Extremophilic EPSs can be applied to nanoparticle technology intwo ways: one is to utilize EPS to form nanoparticles directly, and theother is to use EPS to encapsulate nanoparticles made from anothermaterial. The extremophilic EPSs with negatively charged groups couldperform as polyelectrolytes, which allow them to self-assemble withpositively charged biomolecules to form biodegradable nanoparticlesthrough polyelectrolyte complexation. As a positively charged biopo-lymer, chitosan is often used for polyelectrolyte nanoparticle formationwith negatively charged EPSs (Deepak, Pandian, Sivasubramaniam,Nellaiah, & Sundar, 2015; Karlapudi et al., 2016). Halophilic EPSmauran-chitosan hybrid nanoparticles, produced through an ionic-ge-lation method, manifested stable drug release and biocompatibilitywhen used for antitumor drug encapsulation (Raveendran, Pouloseet al., 2013). Another halophilic EPS levan, produced by Halomonassmyrnensis AAD6T, was investigated for nanoparticle formation throughself-assembly, and its suitability was affirmed as a nanocarrier for de-livery of peptides and proteins (Sezer, Kazak, Öner, & Akbuğa, 2011).

Application of EPSs in the coating or stabilization of chemicallysynthetic nanoparticles also has important potential. Some syntheticnanoparticles possess irreplaceable properties, such as a


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photoluminescence for imaging, but their toxicity in the human bodyneeds to be minimized through suitable modification. Polysaccharidecoated nanoparticles have already been shown to possess rapid uptakeand internalization through the endocytosis effect compared with un-coated nanoparticles, and the cellular toxicity of EPS-coated nano-particles was also notably reduced (Banerjee & Bandopadhyay, 2016).Extremophilic EPSs can therefore be applied as a passivation agent toimprove the biocompatibility of nanoparticles, and make them morefeasible for pharmaceutical applications (Raveendran et al., 2014). Forinstance, quantum dots (QDs) are nanocrystals with a photoluminescentproperty and applied as preferred imaging agents in biological tissuesfor clinical diagnose (Deepagan et al., 2012; Raveendran et al., 2014).However, QDs such as ZnS nanocrystals are synthesized from toxicchemicals in order to maintain their imaging property. These types ofQDs are hydrophobic and water-insoluble, which hinder their applica-tion in the medical field. One solution to overcome these drawbacks isto stabilize QDs with a capping agent, and extremophilic EPSs can be astabilizing agent for QDs to improve their cellular acceptance. In astudy, the stabilization of ZnS-Mn QDs using halophilic EPS mauranwas highly successful in imparting a biocompatible and safe mode ofcellular imaging under in vitro conditions. Anionic EPSs are able to bindwith nanoparticles having positive charge. Additionally, the acetylgroups present in EPSs can bring more positively charged ions to thevicinity of binding sites, thus allowing stronger binding (Raveendranet al., 2014).

4.3.2. EPS-based filmsAdhesive and biocompatible films are an attractive way to offer

fixation to tissues either externally, for wound healing, or internally asa surgical sealant. For the purpose of medical adhesives, such as a burndressing, drug delivery, and implantation, the films are required to keepgood long-term performance on skin or in biological fluids withouttriggering a pathological process (Costa et al., 2013). The inherentfunctions of extremophilic EPSs are to provide adhesion and protectionto bacteria in an extreme environment. Therefore, naturally derivedEPS-based films confer sufficient cohesive strength and maintain abiocompatible response to cells and tissues. The negative charge allowsEPSs to be adsorbed by electrostatic self-assembly and sequential for-mation onto multilayer film. The EPS levan produced by halophilicbacterium Halomonas smyrnensis AAD6T was applied by electrostaticadsorption to construct multilayer film, which demonstrated a pro-mising enhancement of live cell adhesive property. The extremophilicEPS-based film surface, having better biocompatibility, provides a va-luable means to explore novel types of cell-material interactions,leading to an understanding of how to promote or inhibit specific cel-lular responses when contacting bio-based materials (Costa et al.,2013). Moreover, the halophilic EPS levan was mixed with chitosan andpolyethylene oxide (PEO) to produce hybrid films through a solventcasting method. This ternary blend film showed better biocompatiblebehavior compared with chitosan-PEO binary film (Bostan et al., 2014).Another method applied for thin film production using halophilic EPSlevan was matrix-assisted pulsed laser evaporation (MAPLE). This na-nostructured film was also able to sustain cell adhesion and prolifera-tion (Sima et al., 2011).

4.3.3. EPS-based materials through electrospinningElectrospinning is a versatile and relatively cost-effective technique

to fabricate a large variety of soluble or fusible synthetic and naturalpolymers into continuous fibers with diameters in the submicron tonanometer range (Salem, 2007). The electrospinning method has al-ready been applied in many technological fields, and it enables theproduction of novel biomaterials using naturally occurring biopolymerswith complex molecular structures (Torres-Giner, Ocio, & Lagaron,2008). Due to the natural properties of extremophilic EPSs, the mate-rials electrospun from those EPSs are considered sustainable, bio-compatible, biodegradable, and non-toxic. Additionally, the high water-

solubility of EPSs avoids using toxic solvents or additives during elec-trospinning process, making EPS-based electrospun materials excellentcandidates for biomedical engineering applications. However, it can bedifficult to generate neat EPS fibers by electrospinning, since EPS so-lutions tend to have high surface tension, non-ideal viscosity, and ex-cessively strong charge density due to the anionic nature of EPSs(Santos et al., 2014; Torres-Giner et al., 2008). The addition of a hy-drophilic co-polymer as a carrier agent is one way to circumvent thelimitations from those bio-polyelectrolyte (e.g. EPS) solutions(Vashisth, Pruthi, Singh, & Pruthi, 2014). For example, polyvinyl al-cohol (PVA), a biocompatible and water-soluble polymer, can beblended with EPS, reducing the repulsive force from the negativelycharged EPS solution and allowing the generation of uniform nanofi-bers by electrospinning (Qian et al., 2016; Santos et al., 2014; Vashisthet al., 2014). The halophilic EPS mauran was blended with PVA andelectrospun to generate a scaffold with continuous, uniform nanofibers.The mauran-based nanofiber was able to boost cellular adhesion, mi-gration, proliferation, and differentiation of mammalian cells in vitro.The polyanionic nature of extremophilic EPSs increases the negativecharge accumulation on the surface of the scaffold, which is helpful forprotein adsorption and the ability to enhance cellular attachment. Anexcellent property of mauran is that it can keep the same viscosityunder a high concentration of salt and sugar, or under extreme pHvalues; and the stable viscosity of a mauran solution is highly ad-vantageous in obtaining stable electrospinning conditions (Raveendran,Dhandayuthapani et al., 2013). In future studies, those extremophilicEPSs with heavy metal adsorption capability may also be blended withPVA and then electrospun onto a basal microfiltration membrane forwater filtration applications (Santos et al., 2014).

5. Conclusion and prospective

It is now widely considered that extremophilic microorganismsprovide a valuable resource, not only for the elucidation of bioprocessesin extreme environments, but also for the exopolysaccharides theyproduce, which have a valuable range of physicochemical propertiesand highly promising commercial applications. To realize the full valueof these biopolymers, it will be necessary to gain insight into themodulation of EPS biosynthesis in extremophiles. The genome anno-tation and construction of EPS biosynthetic pathways will be of sig-nificant importance in determining the kind of monosaccharide unitsthat can be incorporated into the structures of the EPSs, and how theyare incorporated; as well as how the compositions of monosaccharidesin the EPSs can be affected. Metabolic and genetic engineering willenable the development of effective strategies to successfully enhanceEPS production and engineer EPS properties via tailoring their chemicalcomposition and structure. Applications of extremophilic EPSs in bio-medical, food and pollution-mitigation products can be foreseen basedon their known biodegradability, biocompatibility, non-toxicity, andchemical functionality. It can be anticipated that more extremophilicEPSs from different harsh environments will be discovered, character-ized, and optimized via evolving bioengineering protocols, and thatthey will be employed in high value-added industries with potential forstrong, sustainable growth.

Acknowledgements

This research was supported by the National Science Foundation inthe form of BuG ReMeDEE initiative (Award # 1736255). Authors alsoacknowledge the financial support in the form of CNAM/Bio Centreprovided by the South Dakota Governor’s Office of EconomicDevelopment. Research support from the Department of Chemical andBiological Engineering at the South Dakota School of Mines andTechnology is gratefully acknowledged.


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