ra

H

ins

sede 1

Abstract

1. Introduction

ers because it is economically feasible due to low energyand pressure requirement and high durability of biocatalyst

as compared to catalytic processes (Probstein and Hicks,

subsequently fermented to acetic acid. This process mayachieve higher substrate conversion yield but its produc-tion is very expensive (Parisi, 1989; Vallender and Eriks-son, 1990). Direct conversion of cellulosic biomass toacetic acid by single fermenting organism is economicalbut formed a variety of by-products which include ethanol

* Corresponding author. Tel.: +60 4 599 6417; fax: +60 4 594 1013.E-mail address: chazlina@eng.usm.my (A.H. Kamaruddin).

Available online at www.sciencedirect.com

Bioresource Technology 99 (2Acetic acid is an important industrial feedstock that isproduced mainly from mineral oil and natural gas eitherthrough methanol carbonylation or acetaldehyde oxidation(Spath and Dayton, 2003). At present, high petroleum costdue to substantially depletion of fossil fuel resources hasstimulated the development of new technologies based onrenewable resources. Consequently, fermentation andcatalysis processes that change resource entry from nonre-newable (petroleum) to renewable (biomass) feedstockshave drawn great attention (Klasson et al., 1992). In addi-tion, fermentation process is of great interest for research-

1985). Thus, the focus of many researchers has changedtowards employing acetogenic bacteria as biocatalyst viafermentation process to produce acetic acid almost stoi-chiometrically from renewable resources.

The direct utilization of cheap and abundantly availablebiomass into fermentation process for acetic acid produc-tion includes acid or enzymatic hydrolysis of the cellulosicbiomass to fermentable sugar and followed by bacteria fer-mentation (Slapack et al., 1985). Acid hydrolysis is hin-dered due to low glucose yields and corrosion of theequipment. Enzymatic hydrolysis employs enzymes tobreak down the lignocellulose to fermentable sugars andEorts in optimizing reducing agents, cysteine-HCl H2O and sodium sulde in order to attain satisfactory responses during aceticacid fermentation have been carried out in this study. Cysteine-HCl H2O each with ve concentrations (0.000.50 g/L) was optimizedone at a time and followed by sodium sulde component (0.000.50 g/L). Response surface methodology (RSM) was used to determinethe optimum concentrations of cysteine-HCl H2O and sodium sulde. The statistical analysis showed that the amount of cells producedand eciency in CO conversion were not aected by sodium sulde concentration. However, sodium sulde is required as it does inu-ence the acetic acid production. The optimum reducing agents for acetic acid fermentation was at 0.30 g/L cysteine-HCl H2O andsodium sulde respectively and when operated for 60 h cultivation time resulted in 1.28 g/L acetic acid production and 100% COconversion. 2007 Elsevier Ltd. All rights reserved.

Keywords: Clostridium aceticum; Acetic acid; Synthesis gas; Reducing agents; Statistical analysisOptimization of acetic acid pby chemolithotrophic bacterium

statistical

Jia Huey Sim, Azlina

School of Chemical Engineering, Engineering Campus, Universiti Sa

Received 9 March 2007; received in reviAvailable onlin0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2007.07.004oduction from synthesis gas Clostridium aceticum usingpproach

arun Kamaruddin *

Malaysia, Seri Ampangan, Nibong Tebal, 14300 Penang, Malaysia

form 3 July 2007; accepted 3 July 20073 August 2007

008) 27242735

focus of this work is to optimize three process parameters:

lowering the redox potential of the media. Cultivation

ourcand some lactic acids (Ravinder et al., 2001; Florenzanoand Poulain, 1984). These potential anaerobic bacteriaare Clostridium lentocellum SG6 (Ravinder et al., 2001)and Clostridium thermocellum (Florenzano and Poulain,1984) for a single step fermentation of cellulose to aceticacid.

The employment of microorganism in fermenting CO-containing gas like synthesis gas into chemical productslike acetic acid is another alternatives and ecient route(Klasson et al., 1992). Synthesis gas that was fermentableby bacteria (Natarajan et al., 1998; Reed and Jantzen,1979) could be obtained renewably through the incompletecombustion of biomass and municipal wastes or known asgasication technology (Najafpour et al., 2004). Eubacte-rium limosum KIST 612 (Chang et al., 2001) and Peptostep-tococcus productus U-1 (Vega et al., 1988) are among theacetogenic bacteria that grow and produce acetic acidunder CO gaseous substrate. These bacteria are less favor-able to be used as biocatalysts due to a considerably lowCO tolerance by both bacteria (less than 2.0 atm CO par-tial pressure) (Chang et al., 2001). Clostridium aceticumthat has high CO tolerance (beyond 2.60 atm CO partialpressure), achieve product yield stoichiometrically(0.25 mol acetic acid/mol CO) (Sim et al., 2007) with opti-mum growth in alkaline medium (pH 8.5) lead to its selec-tion as acetic acid producer in this work.

Obligately anaerobic bacteria like C. aceticum, aredened as bacteria which are unable to grow under highredox potential environment. In fermentation media, oxy-gen is primarily responsible for raising the oxidationreduction potential (redox potential Eh) that caused thegrowth inhibition of obligately anaerobic bacteria. The oxi-dationreduction (redox potential Eh) is a measure of thetendency of a solution to be oxidized or reduced (Hungate,1969). Most anaerobic bacteria are inhibited at Eh valueshigher than 100 mV. Reducing agents thus become anessential chemical component in the growth medium as itis responsible to depress and poise the redox potential atoptimum levels. The reducing agents must be nontoxicand at optimum concentration to ensure a satisfactory levelof nal redox potential for the anaerobic organism understudy. The commonly used reducing agents in the anaero-bic cultures includes cysteine-HCl H2O, Dithiothreitol,H2with Palladium Chloride, Na2S 9H2O (Costilow,1981). As in the CO fermentation by strict anaerobic bac-teria like Peptostreptococcus productus, 1.5 mL of sodiumsulde solution was added into fermentation media toensure a low redox potential (Vega et al., 1988). The redoxpotential must be as low as 300 and 360 mV in order toimpose the growth of Clostridium thermoaceticum (Sch-wartz and Keller, 1982). Wieringa (1940) observed that alow redox potential by the addition of 0.1% sodium sulde(Na2S) in the medium was advantageous to the growth ofC. aceticum under CO2and H2 inorganic substrate. There-fore, this study involves the used of reducing agents at opti-

J.H. Sim, A.H. Kamaruddin / Bioresmum conditions to provide a reducing environment toensure C. aceticum growth.medium was accomplished by adjusting the basal mediumto pH 8.5 (optimum pH for C. aceticum) by adding either2 M HCl or 2 M NaOH.

2.2. Batch fermentation start-up

Three independent variables: Cysteine-HCl H2O con-centration, sodium sulde concentration and fermentationtime with three dependent responses: cell concentration,CO residue inside the broth and the acetic acid concentra-tion were utilized in the experimental study. In dening anoptimized reducing agents for acetic acid fermentation, atotal of two sets of experiments were carried out with eachconsisted of one reducing agents: cysteine-HCl H2O or(1) cysteine-HCl H2O, (2) sodium sulde and (3) fermen-tation time in order to enhance maximum acetic acid pro-duction and high CO conversion in batch fermentation.

2. Methods

2.1. Microorganism and cultivation

C. aceticum (DSMZ 1496) in the freezed-dried pelletsform which was obtained from Braunschweig, Germanysculture collection (DSMZ) was used throughout the exper-imental studies. The pellet was rehydrated and the growthwas propagated in the DSMZ recommended growth med-ium 1496. Strain C. aceticum was examined routinely usingmicroscope to check for purity. The chemical compositionsin DSMZ medium 1496 were weighed accordingly for thepreparation of cultivation medium. Dissolved oxygen mustbe eliminated from anaerobic growth medium by degassingthe boiling medium under N2 gas for a few minutes. Serumbottles with N2 headspace were equally lled with 50 mLworking volume and sealed with gas impermeable butylrubber septum-type stoppers and aluminium crimp seals.The liquid media was then autoclaved at 120 C and15 min prior to inoculation. 2.5 mL of 200 g/L fructosesolution instead of H2/CO gaseous substrate was addedas sole carbon source to initiate the dense growth of C.aceticum at the early cultivation process prior to batchstudies. 0.25 mL of sterilized reducing agent was used forFermentation time is another key factor that inuencescell growth in batch system due to depletion in nutrientsources with time and thus aected acetic acid productiondirectly. Therefore, ultimate goal in batch process is toensure maximum acetic acid product to be harvested atoptimum cultivation time. Optimization of processingparameters that included fermentation time for maximumyield of dry cell weight and extracellular polysaccharidecontent produced by the fungus Boletus spp. ACCC50328 were investigated by Wang and Lu (2005). The main

e Technology 99 (2008) 27242735 2725sodium sulde and fermentation time. The employed stud-ied levels for three concerned parameters of cysteine-

ourcHCl H2O, sodium sulde and fermentation time areclearly stated in Sections 2.2.1 and 2.2.2.

Glass serum bottle with an average volume of 163 mLwas used as a reactor for batch fermentation. Experimen-tal medium was prepared approximately in the sameway as cultivation media as described in Section 2.1.The experimental medium compositions were similar asDSMZ medium 1496 except that fructose was being omit-ted and substituted by mixed gas (4% H2:18% Argon:78%CO) as sole carbon source for chemolithotrophic growthof C. aceticum. The serum bottles were ushed with1.80 atm mixed gas or equivalent to 1.40 atm CO partialpressure which acted as carbon source throughout theexperimental studies. The reactors were incubated at30 C and shaken at 200 rpm for 20 min prior to inocula-tion. Ten percent inoculum at exponential phase (v/v)equivalent to 5 mL inoculum discharge from 50 mL work-ing volume were transferred antiseptically to initiate batchfermentation with operating conditions at 30 C and200 rpm. For every changes in the fermentation variable,liquid sample and gas sample were withdrawn from bottlescontinuously for ve days at 12 h time interval. The with-drawn liquid sample was used for cell density measure-ment and acetic acid detection. Two hundred microlitersof gas sample was withdrawn and analysed by gas chro-matograph to determine CO gaseous substrate utilization.All the reactions were performed in duplicates and theresults were reported as mean values. The standard devia-tions for all the experimental results were within 0.05%of the mean values and are not shown due to the smallerror.

2.2.1. Eect of cysteine-HCl H2OThe eect of independent variables: cysteine-HCl H2O

concentration and fermentation time over acetic acid pro-duction were studied. In this experiment, the concentra-tions studied for cysteine-HCl H2O were varied from0.0 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L to 0.5 g/L. Zero concen-tration of cysteine-HCl H2O acted as the control run inthe experiment. The sodium sulde concentration wasmaintained at 0.5 g/L (the concentration used in DSMZmedium 1496) throughout the experiment. The eect ofcysteine-HCl H2O concentration on cell growth, totalCO consumed and acetic acid produced were investigatedfor 120 h cultivation time. Desirable cysteine-HCl H2Othat was quantied from the response surface plot was usedin the experiment of sodium sulde optimization.

2.2.2. Eect of sodium sulde

Sodium sulde and the fermentation time were the twoindependent variables to be optimized while cell concentra-tion, CO uptake and amount of acetic acid produced werethe dependent responses to be maximized. Dierent con-centrations of sodium sulde: 0.0 g/L, 0.2 g/L, 0.3 g/L,0.4 g/L and 0.5 g/L were employed in the work and the

2726 J.H. Sim, A.H. Kamaruddin / Bioreseect towards fermentation parameters was monitoredfor 120 h.2.3. Analytical methods

2.3.1. Cell concentration measurement

A calibration curve consisted of absorbance reading aty-axis against the cell density of C. aceticum at x-axiswas constructed for the purpose of biomass measurement.A half ml of collected liquid sample was diluted to 10-folddilution with distilled water. The solution was measuredfor its optical density by using spectrophotometer at400 nm wavelength. The resulting absorbance readingwas then compared with the generated calibration curveto obtain the corresponding cell concentration (g/L).

2.3.2. Gas measurementIn the study, argon component in the mixed gas acted as

an inert component to calculate the total pressure changesin batch system. The gas compositions were analysed usinggas chromatography (GC) equipped with a thermal con-ductivity detector (TCD). Packed column, Carboxene1000 (Supelco, USA) with dimension of 15 ft 1/8 in wasused for detecting hydrogen, argon and carbon monoxidein the experiment. The injector and detector temperaturewere both at 200 C. The initial oven temperature was40 C, with a rate of 20 C/min until it reached 180 C.Helium (Air Products, Malaysia) was the carrier gas tobe utilized and was set at 30 mL/min owrate. The gas con-centration calculation was done using TotalChrom Work-station Version 6.2 (Perkin Elmer, USA).

2.3.3. Acetic acid measurementSyringe lter with 0.45 lm pore size (Whatman, Eng-

land) was used to lter liquid sample from the cells beforeacetic acid analyses by gas chromatography. The 0.25 mLof ltrate was added with 20 lL of 1% 2-propanol (internalstandard). The solution was then acidied with 30 lL ofpure formic acid for acetic acid analysis. 0.4 lL from themixture was used for gas chromatography analysis whichis equipped with a ame ionization detector (FID). Thecolumn used was Carbopack B-DA/4% Carbowax 20 M(Supelco, USA) with dimensions of 2.0 m length and0.2 cm I.D. The detector and injector temperature wereboth at 225 C. The initial oven temperature was 100 C,with a rate of 10 C/min until it reached 175 C. Helium(Air Products, Malaysia) is the carrier gas with a owrateof 30 mL/min.

2.4. Reducing agents optimization

The responses monitored in the fermentation are: cellconcentration (g/L), CO concentration (mmol) and aceticacid concentration (g/L) resulted from varying concentra-tions of cysteine-HCl H2O and sodium sulde were per-formed graphically to ease the visual evaluation. In orderto obtain the optimum operating conditions, the responsesurface methodology (RSM) was carried out using the

e Technology 99 (2008) 27242735Design-Expert software (version 6.0.6) during the batchstudies.

3. Results and discussion

Section 2.1 in method mentions the steps taken duringstrain propagation and medium preparation for C. aceti-cum cultivation while Section 2.2 is mainly on the start-up process for experimental studies. The pH usuallydecreases during cultivation due to acetogenic activity (ace-tic acid production). The initial pH of medium has been

accordingly with descending cysteine-HCl H2O concentra-tion. During exponential phase of growth, the biomass pro-

The curves for CO consumption rate as a function of timeat various cysteine-HCl H2O concentration were of simi-lar quantitative trend that resemble a bell shape.

In general, the production of acetic acid at each concen-tration of cysteine-HCl H2O does not dier signicantlyfrom each other (Fig. 3). This means that cysteine-HCl H2O with concentrations of 0.00.5 g/L did not exertsignicant eect to the acetic acid production. Within 84 hfermentation time, the acetic acid production was the high-est at 0.3 g/L cysteine-HCl H2O compared to other con-centrations. Although cells in 0.5 g/L cysteine-HCl H2Oreached the highest peak of acetic acid concentration of2.35 g/L at 108 h (Table 2) but the acetic acid productionbefore 108 h was mostly maintained at low concentrationcompared to other cysteine-HCl H2O concentrations(Fig. 3). Therefore, 0.3 g/L cysteine-HCl H2O was thedesirable concentration to be applied in the batch systemsince it maintained a relatively high level of cell concentra-

J.H. Sim, A.H. Kamaruddin / Bioresourcduced was inversely proportional to the cysteine-HCl H2Oconcentration. High cysteine-HCl H2O concentration,0.5 g/L was less favorable to the cell growth which leadto the lowest cell concentration, 0.49 g/L. Therefore, theinhibition of cysteine-HCl H2O to the cell growth waslikely to occur at 0.5 g/L as indicated by apparently lowestcell growth curve in Fig. 1. In other words, 0.00.4 g/L ofcysteine-HCl H2O concentration were sucient to reduce

Table 1pH of medium at various cysteine-HCl H2O and sodium suldeconcentration during initial and nal fermentation time

Cysteine (g/L) 0.0 0.2 0.3 0.4 0.5

pH medium without cysteine at 0 h 8.98 8.84 8.96 8.64 8.75pH medium at 120 h 7.08 7.13 7.13 7.18 7.23

Sodium sulde (g/L) 0.0 0.2 0.3 0.4 0.5adjusted to optimum pH 8.5, whereas the nal pH after120 h at various cysteine and sodium sulde concentrationswere recorded and shown in Table 1. The nal pH recordedat dierent cysteine and sodium sulde concentrations weresimilar, ranging between pH 7.087.27. This indicated thatall experiments were subjected to small pH variationsthroughout the experimental study, thus any variation inresponses due to pH can be neglected.

3.1. Eect of cysteine-HCl H2O on acetic acid fermentation

Cysteine-HCl H2O complements sodium sulde as thereducing agents for anaerobic media. Reducing agent inappropriate concentration is fairly important to anaerobicbacteria so as to poise and depress the redox potential inmedium to the levels that would initiate cell growth. Theexperiment was conducted with the aim to obtain the opti-mum concentration of cysteine-HCl H2O and fermenta-tion time through RSM.

Fig. 1 presents the cell growth in dierent concentrationsof cysteine-HCl H2O as a function of time. All cells wereexperiencing the same and typical sigmoid growth curvewhere lag phase occurred for the initial 12 h incubationperiod. Eventually, there appeared to be small variationwithin the maximum cells concentration achieved at eachlevels of cysteine-HCl H2O (Table 2). The specic growthrate as indicated by the steepness of the slope increasedpH medium without sodium at 0 h 8.80 8.59 8.93 8.70 8.66pH medium at 120 h 7.11 7.27 7.13 7.09 7.20the redox potential to the extend that would permit anaer-obic cell growth.

The use of cysteine-HCl H2O ranging between 0.0 and0.4 g/L resulted in minimum CO residue inside the system.After 48 h of incubation period, two regions were clearlydened based on the rapidity in the amount of CO con-sumed. Cysteine-HCl H2O of 0.5 g/L with low CO con-sumption rate was situated at higher region while otherswith similarly high CO consumption rate gathered at lowerregion. CO uptake rate at 0.5 g/L cysteine-HCl H2Odecreased sharply after 48 h as compared to other cys-teine-HCl H2O concentration and the CO consumptiongradually retarded (Fig. 2). The total CO consumed at0.5 g/L cysteine-HCl H2O was 4.97 mmol whereas thetotal CO uptake at other cysteine-HCl H2O concentra-tions were between 5.77 and 6.29 mmol (Table 2). FromFig. 2 and Table 3, the CO consumption rates attainedby all concentrations of cysteine-HCl H2O (ranging from2.53 to 3.19 mmol/L h) were not signicantly dierent.

Time, h0 20 40 60 80 100 120 140

Cell c

once

ntra

tion,

g/ L

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.0 g/ L0.2 g/ L0.3 g/ L0.4 g/ L0.5 g/ L

Cysteine-HCl.H2O Concentration

Fig. 1. Eect of cysteine-HCl H2O on cell concentration.

e Technology 99 (2008) 27242735 2727tion throughout the experimental study and achievednearly 100% CO conversion.

Table 2Maximum cell concentration, CO uptake and acetic acid produced under dierent cysteine-HCl H2O and sodium sulde concentration

Reducing agents(g/L)

Cell concentration CO concentration Acetic acid concentration

Maximum produced(g/L)

Fermentationtime (h)

Maximum consumed(mmol)

Fermentationtime (h)

Maximum produced(g/L)

Fermentationtime (h)

Cysteine-HCl H2O0.0 0.56 48 6.29 120 1.52 1080.2 0.55 48 5.82 120 1.52 1080.3 0.59 60 6.04 120 2.08 1080.4 0.51 48 5.77 120 1.87 1200.5 0.49 48 4.97 120 2.35 108

Sodium sulde

0.0 0.81 36 6.45 48 1.42 1200.2 0.78 36 6.45 48 1.72 600.3 0.80 36 6.41 48 1.89 600.4 0.71 48 6.44 60 1.46 1200.5 0.79 36 6.43 60 1.43 108

Time, h0 12 24 36 48 60 72 84 96 108 120 132

CO c

onsu

mpt

ion

rate

, mm

oles

/L.h

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0.0 g/L0.2 g/L0.3 g/L0.4 g/L0.5 g/L

Cysteine-HCl.H2O Concentration

Fig. 2. Eect of cysteine-HCl H2O on CO consumption rate.

Table 3Comparison of fermentation parameters under dierent cysteine-HCl H2O and sodium sulde concentration

Reducingagents (g/L)

CO Acetic acid Acetic acid yield

Consumption rate(mmol/L h)

Fermentationa

time (h)Formation rate(g/L h)

Fermentationa

time (h)Yp/s Fermentation

a

time (h)Yp/x Fermentation

a

time (h)

Cysteine-HCl H2O0.0 3.19 24 0.041 48 0.31 12 2.33 1080.2 2.62 24 0.033 48 0.61 12 2.59 1080.3 2.82 36 0.070 108 0.69 12 3.43 1080.4 2.92 36 0.051 36 0.73 12 3.29 1200.5 2.53 36 0.112 108 1.06 12 5.26 108

Sodium sulde

0.0 5.76 24 0.053 48 0.18 120 2.63 1200.2 5.37 24 0.100 48 0.18 96 1.83 960.3 5.14 24 0.056 60 0.25 60 2.60 1200.4 4.84 24 0.058 24 0.19 120 2.61 1200.5 5.29 24 0.048 24 0.19 108 2.36 108

a Fermentation time reported was referring to time consumed for maximum CO consumption rate, maximum acetic acid formation rate and maximumacetic acid yield, respectively.

Time, h0 12 24 36 48 60 72 84 96 108 120 132

Acet

ic a

cid

conc

entra

tion,

g/L

0.0

0.5

1.0

1.5

2.0

2.5

0.0 g/L0.2 g/L0.3 g/L0.4 g/L0.5 g/L

Cysteine-HCl.H2O Concentration

Fig. 3. Eect of cysteine-HCl H2O on acetic acid concentration.

2728 J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735

3.1.1. Response surface method (RSM) analysis

The relationship between responses: cell concentrationand acetic acid concentration with factors: cysteine-HCl H2O and fermentation time were well tted withquadratic model as expressed by Eqs. (1) and (2) whilethe CO residue response can be predicted by cubic

model as in Eq. (3). The CO concentration and aceticacid concentration were transformed to square root inorder to solve the abnormal response problems. Empiricalmodels were in good t to the experimental results (R2

ranging from 0.84 to 0.97, data not shown) for all theresponses

Cysteine-HCl.H2O (g/L)Fe

rmen

tatio

n Ti

me

(h)0.00 0.13 0.25 0.38 0.50

0

30

60

90

120

0.020.13

0.25

0.36

0.36

0.48

0.25

2.04

3.82

5.61

7.40

CO

con

cent

ratio

n (m

moles

)

0.00

0.13

0.25

0.38

0.50

0306090

120

Cysteine-HCl.H2O(g/L)

Fermentation Time (h)

Cysteine-HCl.H2O(g/L)

Fermentation Time (h)

Cysteine-HCl.H2O (g/L)

Ferm

enta

tion

Tim

e (h)

0.00 0.13 0.25 0.38 0.500

30

60

90

120

0.87

1.24

1.61

1.982.352.74

3.37

0.02

0.41

0.80

1.18

1.57

d co

ncen

tratio

n (g/

L)

ine-

HCl.H

2O (g

/L)

0.25

0.38

0.50

0.38

0.56

0.73

0.91

1.09

0.22

-0.10

0.07

0.25

0.42

0.59

Ce

ll con

cent

ratio

n (g

/L)

0.00

0.13

0.25

0.38

0.50

0 30 60 90

120

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735 2729Cysteine-HCl.H2O(g/L)

Ac

etic

aci

030

6090

120

0.00

0.13

0.25

0.38

0.50

Fermentation Time (h) Fig. 4. Response surface plot and contour plot on cysteine-HCl H2O and feproduced.Fermentation Time (h)

Cyst

e

0 30 60 90 1200.00

0.13rmentation time for (a) cell concentration; (b) CO residue; (c) acetic acid

Cell concentration 0:017528 0:23341 Cysteine 0:015248 Fermentation time 1:00676E 004 Fermentation time2; 1

SqrtAcetic acid concentration 0:02 0:20233 0:022475 Fermentation time 1:18977E 004 Fermentation time2; 2

SqrtCO concentration 2:66915 1:32369 Cysteine 0:028229 Fermentation time 9:70078 Cysteine2 8:47015E 005 Fermentation time2 0:010704 Cysteine Fermentation time 14:49455 Cysteine3:

3

The response surface for cell concentration is presented inFig. 4a. The eect of cysteine-HCl H2O on cell growthwas less pronounced during early fermentation time (lessthan 30 h). However, after 45 h of fermentation, the cellsin higher cysteine-HCl H2O concentration took longertime to achieve 0.48 g/L cell concentration as clearly seenfrom the response surface plot in Fig. 4a. The same quan-titative trend for cell concentration response surface plot(Fig. 4a) occurred in the response surface plot of CO con-centration in Fig. 4b. The eect of cysteine-HCl H2O onthe CO uptake by C. aceticum was becoming signicantwhen incubated for more than 30 h. Approximately 90%of CO conversion was located at around 90 h fermentationtime over a wide range of cysteine-HCl H2O. Fermenta-tion time was the key parameter for acetic acid production(Fig. 4c). Long fermentation time (for more than 50 h) wasnecessary for higher acetic acid production by C. aceticum

Table 4Analysis of variance (ANOVA) for the regression model and the respective model terms on the studies of cysteine-HCl H2O and sodium suldeconcentration

Source Model terms Sum of squares Mean square F Value Prob > F Remarks

Cysteine-HCl H2OCell concentrationQuadratic 1.69 0.56 81.49

over the entire cysteine-HCl H2O concentration. As statedearlier, cysteine-HCl H2O concentration seems to exertminor eect on acetic acid productivity from the graphicalpresentation of acetic acid production as a function oftime. However, the signicance test performed during AN-OVA on cysteine-HCl H2O to acetic acid concentrationresponse shows that (Prob > F) far exceeds 0.05 (Table4). This means that the eect from cysteine-HCl H2Oalone on acetic acid produced was insignicant to beincluded in the empirical model for process optimization.Therefore, cysteine-HCl H2O alone was insignicant onthe acetic acid concentration. It can be concluded thatthe eect of cysteine-HCl H2O (A) to acetic acid producedwas insignicant while fermentation time (B) remain to bethe key factor for the three responses (Table 4).

3.1.1.1. System optimization within designated constraints.

The objectives of the study were to maximize cell concen-tration and acetic acid produced with minimum CO residue

left inside the system (Table 5). Therefore, the most desir-ability experimental region was found at 0.30 g/L cys-teine-HCl H2O and at 60 h (Fig. 5). Thus, when thesystem was operating under the optimum conditions, theCO residue was 1.71 mmol and the acetic acid producedwas 1.20 g/L as compared to the predicted values of1.93 mmol CO concentration and 1.23 g/L acetic acid con-centration. The cysteine-HCl H2O concentration was saidto be successfully reduced from initial 0.5 g/L to 0.3 g/Lwhile still retaining high acetic acid productions.

3.2. Eect of sodium sulde on acetic acid fermentation

Sodium sulde as mentioned earlier in Section 3.1, ispart of the reducing agent that is responsible in initiatingthe growth for anaerobic bacteria. Sodium sulde is thenal component chosen to be minimized while cysteine-HCl H2O were constant at its optimum concentrations.

Table 5The preset goal with the constraints for all the independent factors and responses in numerical optimization

Variables Ultimate goal Experimental region

Lower limit Upper limit

Eect of cysteine-HCl H2OFactor Cysteine-HCl H2O, A (g/L) In range 0.00 0.50

Fermentation time, B (h) Minimized 0 120

Response Cell concentration (g/L) In range 0.00 0.69CO concentration (mmol) Minimized 0.16 6.46Acetic acid concentration (g/L) Maximized 0.024 5.51

Eect of sodium sulde

Factor Na2S 9H2O, A (g/L) In range 0.00 0.50M

IMM

0

) 90

0.351

0.439

J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735 27310.000

0.132

0.263

0.395

0.526

D

esira

bility

0.00 0.13

0.25 0.38

0.5

030

60 90

120

Cysteine-HCl.H2O(g/L)

Fermentation Time (h) Fermentation time, B (h)

Response Cell concentration (g/L)CO concentration (mmol)Acetic acid concentration (g/L)Fig. 5. Response surface plot of the desirability regionCysteine-HCl.H O (g/L)

Ferm

enta

tion

Tim

e (h

0.00 0.13 0.25 0.38 0.500

30

60

0.088 0.1750.263

0.351

0.439

0.5060.506inimized 0 120

n range 0.00 0.81inimized 0.00 6.45aximized 0.71 1.89

1200.1750.2632

across cysteine-HCl H2O and fermentation time.

The desirable level of sodium sulde was identied byresponse surface method analysis (RSM).

Generally, cells in 0.0 g/L and 0.5 g/L sodium suldepossesses the highest and second highest cell growth raterespectively while cells replicated at about the same ratein the mid studied range of sodium sulde (0.2 g/L, 0.3 g/L and 0.4 g/L). In addition, cells cultured at this range ofsodium sulde (0.2 g/L, 0.3 g/L and 0.4 g/L) exhibitedgrowth curve with lag phase for the rst 12 h before enter-ing exponential phase. As seen from Table 2, the cell con-centration reached the peak values of 0.780.81 g/L within36 h fermentation time throughout the whole range ofsodium sulde except for 0.4 g/L. The lowest cell concen-tration i.e. 0.71 g/L was obtained at 0.4 g/L sodium sulde.Apparently, sodium sulde when employed at high concen-tration (0.5 g/L) did not show toxicity to the cell growth.

2732 J.H. Sim, A.H. Kamaruddin / BioresourcThe sodium sulde within the studied range was sucientto depress and poise the redox potential of the medium thatallowed the anaerobic bacteria propagation.

It was interesting to note that the CO reduction for allthe sodium sulde levels were nearly of the same rate.Low sodium sulde concentration (0.0 g/L, 0.2 g/L and0.3 g/L) tends to achieve 100% CO conversion at shorterfermentation time, 48 h as compared to high sodium sulde.In addition, the CO consumption rate increased with adecrease in sodium sulde concentration except at 0.5 g/Lsodium sulde (Table 3). The maximum CO being con-sumed and maximum CO consumption rate achieved atvarying concentration of sodium sulde were almost similaras clearly observed from Tables 2 and 3. Therefore, theapplication of sodium sulde within the studied range inbatch system encourages 100% CO uptake by C. aceticum.

Although sodium sulde did not seem to exert signicanteects on cell growth and CO uptake rate, but sodium sul-de concentration aects appreciably the acetic acid pro-duction (Fig. 6). At 0 h fermentation, the concentration ofacetic acid which were close to 1 g/L in all cases resultedfrom the transfer during inoculation process. Acetic acidwas initially present in relatively high concentrations due

0 12 24 36 48 60 72 84 96 108 120 132

Acet

ic a

cid

conc

entra

tion,

g/L

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.0 g/ L0.2 g/ L0.3 g/ L0.4 g/ L0.5 g/ L

Na2S.9H2O ConcentrationTime, h

Fig. 6. Eect of sodium sulde on acetic acid concentration.concentration to the key factors (Eqs. (5) and (6)). Cellconcentration was transformed to square root scale whileCO concentration was transformed to natural log scaleand resulting in relatively high R2, 0.9492 and 0.9224,respectively (data not shown).

SqrtCell concentration 0:01 0:085147 0:038915 Fermentation time 5:75372E 004 Fermentation time2 2:42571E 006 Fermentation time3; 4

Ln CO concentration 0:06 2:56819 0:11276 Fermentation time 5:76131E 004 Fermentation time2; 5

Acetic acid concentration 0:13921 0:64369Na2S 9H2O 0:030659 Fermentation time 1:47339E 004 Fermentation time2: 6

The impact of sodium sulde on cell growth and CO up-take were completely eliminated as clearly observed fromthe response surface in Fig. 7a and b. In other words, cellconcentration and CO concentration at xed fermentationtime can be determined at any point along the line or fer-to the preculture production from inoculum broth. Sodiumsulde at 0.3 g/L produced the highest net acetic acid con-centration, 1.89 g/L in shortest fermentation time i.e. 60 hcompared to others as shown in Table 2. Table 3 summa-rizes the maximum substrate uptake rate, maximum pro-duction rate and maximum product yield under dierentsodium sulde concentration to ease the investigation ofthe sodium sulde eect. C. aceticum in 0.3 g/L sodium sul-de fermented CO stoichiometrically to acetic acid with 4 gof CO completely converted to 1 g of acetic acid as suggestedin the stoichiometric reaction (Eq. 1) and resulted in0.25 gacetic acid/gCO product yield,Yp/s (Table 3). In contrast,sodium sulde at concentrations higher or less than 0.3 g/Lwere inhibitingC. aceticum to convert CO gaseous substrateto acetic acid and caused low production yield of 7576% orYp/s = 0.180.19 compared to 100% product conversion(Yp/s = 0.25) being achieved at 0.3 g/L. The eciency of cellin producing acetic acid which was denoted by Yp/x (2.362.63) were less inuenced by the applied sodium sulde con-centrations except at 0.2 g/L. Therefore, 0.3 g/L sodium sul-de was the most suitable concentration to be applied in thefermentationmedium that resulted inmaximum cell concen-tration, complete CO conversion (100%) andmaximum pro-duction within short cultivation time (Table 2).

3.2.1. Response surface method (RSM) analysis

Eqs. (4)(6) were the empirical models used for generat-ing response surface plot. Cell concentration was best pre-sented with cubic model (Eq. (4)) while quadratic modelwas used to correlate CO concentration and acetic acid

e Technology 99 (2008) 27242735mentation time is the key factor for both responses. There-fore, acetic acid fermentation has to be cultivated within

optimum period in order to achieve dense cell at late expo-nential phase and targeted to zero CO residue. However,the amount of sodium sulde used in the study exertedminor inuence on the acetic acid produced as clearlyviewed from Fig. 7c. Acetic acid production was said tobe proportional to the length of the fermentation timebut inversely proportional to the applied sodium sulde

during batch fermentation as clearly viewed from Fig. 7c.In others word, acetic acid production was preferably en-hanced at reduced amount of sodium sulde concentrationbut longer incubation time. The maximum acetic acid pro-duction of 1.14 g/L was presumed to be occurring at mod-erate fermentation time over wide range of sodium suldebut less than 0.50 g/L.

-0.00

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S.9H

2O (g

/L)

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S.9H

2O (g

/L)

Fermentation time (h)0 30 60 90 120

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J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735 2733Na2S.9H2O (g/L)

-0.46

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030

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Fermentation time (h)Fig. 7. Response surface plot and contour plot on sodium sulde and fermentatNa S.9H O (g/L)

Ferm

enta

t

0.00 0.13 0.25 0.38 0.500

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ion time for (a) cell concentration; (b) CO residue; (c) acetic acid produced.

50

regi

ourc3.2.1.1. System optimization within designated constraints.

The optimum working conditions for all responses is givenin Table 5. The most desirable operating conditions thatoptimized all the responses simultaneously were locatedat 0.30 g/L sodium sulde and at 60 h (Fig. 8). The amountof CO residue was 0.0 mmol and the acetic acid being pro-duced in the system was 1.28 g/L. The predicted resultswere 0.0 mmol CO concentration and 1.17 g/L acetic acidconcentration. Therefore, the optimum reductants for C.aceticum were at 0.3 g/L each for cysteine-HCl H2O andsodium sulde. 0.3 g/L were proven again as optimumreductants when drastic improvement of 89% in specicgrowth rate, l was observed in optimum medium com-pared to medium DSMZ 1496. The specic growth rate,l for medium DSMZ 1496 before and after process optimi-zation of reductants was 0.0326 h1 and 0.0617 h1,respectively.

4. Conclusion

0.000

0.170

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esira

bility

0.000.13

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0.

0

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Na2S.9H2O (g/L) Fermentation time (h)

Fig. 8. Response surface plot of the desirability

2734 J.H. Sim, A.H. Kamaruddin / BioresBoth reducing agents have shown to be signicantlyaecting the acetic acid fermentation by C. aceticum. Nor-mal graphical plot and response surface plot illustratedthat inhibition of cysteine-HCl H2O over cell concentra-tion and the CO uptake ability existed when high cys-teine-HCl H2O concentration was employed (0.5 g/L orabove). In contrast to cysteine-HCl H2O, sodium suldeon its own did exert certain eect on acetic acid producedbut has no inuenced on cell concentration and CO uptakebased on the 2-D and 3-D surface plot. Optimum operat-ing conditions were found to be at 0.3 g/L for both cys-teine-HCl H2O and sodium sulde and this study hassuccessfully reduced 40% of the initial reducing agentsemployed, from 0.5 g/L (concentration suggested inDSMZ growth media) to 0.3 g/L. The optimum operatingregion which corresponded to 0.3 g/L cysteine-HCl H2Oand 0.3 g/L sodium sulde for 60 h fermentation time pro-duced 1.28 g/L acetic acid and attained 100% COconversion.References

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The present research was made possible through anIRPA grant project (01-02-05-32230EA011) sponsored byMinistry of Science, Technology and Innovations (MOS-TI), Malaysia and graduate assistant scheme allowanceawarded by Universiti Sains Malaysia (USM). Dr. Habi-bollah Younesi and Dr. Long Wei Sing are acknowledgedfor their assistance and comments.

Na2S.9H2O (g/L)

Ferm

enta

tion

time

(h)

0.00 0.13 0.25 0.38 0.500

30

60

90

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0.113 0.226

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J.H. Sim, A.H. Kamaruddin / Bioresource Technology 99 (2008) 27242735 2735

Optimization of acetic acid production from synthesis gas by chemolithotrophic bacterium - Clostridium aceticum using statistical approachIntroductionMethodsMicroorganism and cultivationBatch fermentation start-upEffect of cysteine-HCl middot H2OEffect of sodium sulfide

Analytical methodsCell concentration measurementGas measurementAcetic acid measurement

Reducing agents optimization

Results and discussionEffect of cysteine-HCl middot H2O on acetic acid fermentationResponse surface method (RSM) analysisSystem optimization within designated constraints

Effect of sodium sulfide on acetic acid fermentationResponse surface method (RSM) analysisSystem optimization within designated constraints

ConclusionAcknowledgementsReferences