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Acetic Acid Batch Fermentation Process by Clostridium Thermoaceticum Harumi Veny, Masitah Hasan my_harumi@yahoo.com, masitahhasan@um.edu.my Department of Chemical Engineering Faculty

Acetic Acid Batch Fermentation Process by

Clostridium Thermoaceticum

Harumi Veny, Masitah Hasan my_harumi@yahoo.com, masitahhasan@um.edu.my

Department of Chemical Engineering Faculty of Engineering

University of Malaya

ABSTRACT

Acetic acid was produced through

anaerobic batch fermentation process by using

Clostridium thermoaceticum as microorganism

The kinetic parameters (i.e µmax , Y p/s, Y x/s ) and

the relationship between growth and acetic acid

product formation were determined for all

environmental effects studied in batch

fermentation to establish the optimum condition

for this system The optimum condition obtained

from this batch fermentation studies was at pH 6

and temperature of 60 o C with the maximum

product yield (Y p/s ) of 0.88 g acetic acid /g

glucose and maximum cell yield (Y x/s ) of 0.11 g

cell/g glucose The maximum specific growth

rate was 0.11 hour -1 achieved at initial glucose

concentration of 30 g/L The acetic acid

fermentation profile from these studies showed a

mix of growth associated and non-growth

associated

Keywords: Acetic acid, Clostridium

thermoaceticum, Single step anaerobic

fermentation

(1) INTRODUCTION

Presently, almost all acetic acid in chemical

industry products is produced synthetically

About sixty two percent is produced by the

carbonylation of methanol and the rest by the

liquid phase oxidation on n-butane and direct

oxidation of acetaldehyde (Wacker process) [1]

The major disadvantages of the synthetic process

are the need of high temperature and pressure, a good agitation, the threat of explosion, the high cost of catalyst, and the dependence on non-renewable sources

of raw material (crude oil) Hence, acetic acid productions to be carried out by biological (fermentation) mean become more attractive The production of acetic acid via fermentation by using renewable biomass feedstock has also been studied as alternatives to acetic acid production from petroleum or natural gas feedstock [2] The fermentation process could reduce the high cost since it uses renewable resource as raw material and low energy utilization The problem with the conventional fermentation process is that the two step aerobic fermentation could only produce low acetic acid product yield (around 0.67 g acetic acid/g glucose) This process also involved two stages for converting glucose into ethanol followed by oxidation of the ethanol to acetic acid Hence, it needs two separate fermentors A possible solution to this problem is a single step anaerobic fermentation which

is described in this paper

The single step anaerobic process requires the absence of oxygen; hence the condition during all fermentations should be strictly anaerobic by gassing with CO2 The single step anaerobic fermentation process has a higher product yield, could eliminate separate fermentors for the different stages of acetic acid production and reduces the time required In these experiments, the single step anaerobic fermentation

process uses Clostridium thermoaceticum which

converts glucose almost quantitatively to the theoretical amount of acetic acid The reaction for this process is as follows:

C6H12O6 3 CH3COOH

The acetic acid product yield from this

single step fermentation process from several

studies is around 0.80-0.90 gram acetic acid per

gram of glucose [4,5,9] The maximum specific

growth rate (µmax) from previous researchers

[4,5,6,8] was 0.1 h-1 to 0.14 h-1 in batch

fermentation system

(2) MATERIAL AND METHODS

Microorganism

Clostridium thermoaceticum bacteria from

DSMZ, Germany was used in this experiment

Media and growth condition

RCM and a modification of DSMZ

recommended medium

RCM medium is used for maintenance and stock

cultures in solid and semi solid form, which then

is sub cultured for every 2 weeks The RCM

composition is: Yeast extract (3 g/l), Lab–Lemco

Powder (10 g/l), Peptone (10 g/l), Glucose ( 5

g/l), Soluble starch (1 g/l), Sodium Chloride

(5g/l), Sodium acetate (3 g/l), Cysteine

hydrochloride (0.5 g/l), Agar (0.5 g/l) or (15 g/l

for solid medium)

Medium for cultivation of Clostridium

thermoaceticum is a modification of some

medium compositions from earlier researches [4,

5,10] The compositions are:

Solution A: Glucose (variable), Water (150)

Solution B: Yeast extract Difco(5 g), Tryptone

difco(5 g),Sodium acetate (1.55 g) (NH4)2SO4 (1

g), MgSO4 7 H2O (0.25 g),Fe(NH4)2(SO4)2 6

H2O (0.039 g), Co(NO3)2 6 H2O (0.03 g),

Na2WO4 2H2O (3.30 mg), Na2MoO4 2H2O

(2.4 mg), ZnCl2 (1.4 mg),Na2SeO3 5H2O(0.2

mg), NiCl2 6H2O (0.2 mg), Resazurin 0.1% (1

ml), Distilled water (550 ml), Cysteine

hydrochloride (0.25 g), Na2S.9H2O 5%(5 ml)

Solution C: NaHCO3 (16.8 g), K2HPO4 (7 g),

KH2PO4 (5.5 g), Distilled water (300 ml)

The media was prepared by heating before

sterilization, to help driven off dissolved oxygen

Growth condition, medium and inoculums

preparation, and culture storage condition have

been previously described [3]

Analytical methods

Acetic acid production was analyzed using Gas

chromatograph Growth of Clostridium thermoaceticum

was monitored spectrophotometrically by measuring the optical density at 600 nm A standard curve relating the optical density to cell dry weight was constructed The glucose utilization was analyzed by DNS method spectrophotometrically at optical density of 550 nm Detail analytical methods have been previously described[3]

Batch fermentation studies

The batch fermentation studies were carried out in 2.5 L KO biotech fermentor with a working volume of 1.5 L The inoculums for all batch fermentation runs were incubated for 48 hours at 60oC The anaerobic condition for fermentation process was maintained by continuous supply of sterile CO2 gas The pH was maintained by automatic addition of 2N KOH and 2N HCl The foam formed during the process was reduced

by automatic addition of antifoam agent and with the help of foam breaker in the stirrer The stirrer rate for all batch fermentation was maintained at a low rate of

50 rpm

(3) RESULTS AND DISCUSSIONS

The environmental effects on batch fermentations were studied by varying the pH (i.e pH 5 to 7) and initial glucose concentration (15 to 60 g/L) The environmental effect studies achieved the maximum acetic acid yield of 0.88 g/g and growth yield of 0.1 g/g

at pH 6 and 60oC This is probably because of the pH optimum conditions for the bacteria growth is near to

neutral pH while the pH of acetic acid production was

expected to be at acid pH See Figure 1 According to literature[8], at low pH below pH 6 undissociated acetic acid is responsible for growth inhibition and at high pH (above pH 6) ionized acetate is responsible for growth inhibition. The maximum specific growth rate (µmax) obtained was 0.11 h-1 at initial glucose concentration of

30 g/L The specific growth rate increased with increase

in initial glucose concentration from 15 g/L to 30 g/L before it tends to decrease up to concentration of 60 g/L See Figure 2.This may be so as the substrate concentration has exceeded the substrate critical concentration (S>Scrit)[7] The maximum specific

growth rates were determined at the exponential

phase and through graphical data of plot ln(x/xo)

as a function of time (t) by following the growth

rate equation:

X

dt

dx

.

µ

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

pH

0 0.02 0.04 0.06 0.08 0.1 0.12

Product Yield Growth yield

Fig.1 Acetic acid yield and growth yield

at different pH

0

0.02

0.04

0.06

0.08

0.1

0.12

Initial glucose concentration (g/L)

Fig.2 The maximum specific growth rate

The maximum kinetic parameters result at

the optimum condition are given in Table 1

Table.1 Kinetic parameters Initial glucose

concentration Temp pH

µ max (h-1) Y(g.gx/s -1) Y(g.gp/s-1)

The batch fermentation profile at optimum condition of pH 6 and 60oC from this study is given in Figure 3 This fermentation profile showed that the acetic acid concentration increased with the increase in biomass/dry cell However, the acetic acid still increased even when the dry cell had already ceased to grow This phenomenon illustrates a mixed growths associated which also have been reported previously in some literature [6, 8]

0 2 4 6 8 10 12 14 16

0 20 40 60 80 100 120 140

Time (hours)

0 2.5 5 7.5 10

acetic acid glucose dry weight

Fig.3 Batch fermentation profile at pH 6 and 60 o C

[4] Conclusion

The conclusions which could drawn from this acetic acid batch fermentation process from glucose by

Clostridium thermoaceticum are:

1 Overall the optimum condition for acetic acid production obtained from these studies was at pH 6 and temperature of 60oC

2 The maximum production yield (Yp/s) achieved was 0.88 g of acetic acid/g glucose which is within the range of earlier reported researches on this process

Y p/s

Y x/s

3 The maximum biomass yield (Yx/s) of 0.11 g

biomass/g glucose and maximum specific

growth rate (µmax) of 0.11 hour-1 were obtained

at initial glucose concentration of 30 g/L

4 The relation between the growth and acetic

acid production from these studies showed a

mix of non growth and growth associated

where the acetic acid was still produced even

when growth has ceased

[5] References

[1]Busche, R.M.(1991) Extractive Fermentation

of acetic acid; Economix trade off between yield

of Clostridium and concentration of Acetobacter

Appl.Biochem.Biotech.28(29):605-621

[2] Cheryan,M., Parekh,S., Shah,M.,

Witjitra,K.,(1997) Production of acetic acid by

Clostridium thermoaceticum

Adv.Appl.Microbial.V.43.pp.1-31

[3] Harumi.V,

Masitah.H,Ramachandran.K.B,.(2003) “Acetic

acid production by single step anaerobic

fermentation of glucose In; Proceeding of

Technical Postgarduate Symposium 2003

(TECHPOS’03)

[4] Ljungdahl,L.G., (1983).Formation of acetate

using homoacetate fermenting anaerobic

bacteria In: Organic chemicals from biomass,

edited by Wise,D.L., pp.219 The

Benjamin/Cummings Publishing Company,Inc

[5]Parekh,S.R., Cheryan,M., (1990) Acetate

Production from Glucose by Clostridium

thermoaceticum Process Biochemistry

International.117-121

[6] Parekh,S.R., Cheryan.M., (1991) Production

of acetate by mutant strain of Clostridium

thermoaceticum.Appl.Microbiol.Biotechnol.36:3

84-387

[7] Sugaya,K., Tuse,D., Jones,J.L., 1986

Production of acetic acid by Clostridium

thermoaceticum in batch and continous

fermentations Biotech.Bioeng v.xxviii.pp.678-683 [8] Pirt, S.John, (1975) Principles of microbe and cell cultivation, Halsteed Press Book.UK

[9] Wang,I.C.D., Fleishchaker.J., Wang,Y.G., (1978) A novel route to the production of acetic acid by

fermentation AIChE Symposium series, No.181, vol

74

[10] Witjitra,K., Shah,M.M., Cheryan,M., (1996) Effect of nutrient sources on growth and acetate

production by Clostridium thermoaceticum Enzyme &

Microbiol.Technol.19:322-327