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Heterofermentation of Clostridium thermolacticum 63 ___________________________________________________________________________ 4 HETEROFERMENTATION OF CLOSTRIDIUM THERMOLACTICUM AND ITS FREE-CELL C O-CULTURE WITH MOORELLA THERMOAUTOTROPHICA 4.1 Summary The metabolites of lactose fermentation by a pure culture of Clostridium thermolacticum were lactate, acetate, ethanol, carbon dioxide and hydrogen at thermophilic temperature (60°C) and at pHs between 5.8 and 8.5. However, there was a metabolic shift from heterolactic to homolactic pathway during the fermentation, depending on the growth and pH conditions. Production of acetate and ethanol were growth associated, with an acetate to ethanol ratio of 1:1, carbon dioxide and hydrogen were formed throughout the fermentation, while lactate was produced only at the end of the exponential growth phase and during the stationary phase at a lower pH with a yield close to the maximum of 4 mol per mol lactose. C. thermolacticum was strongly inhibited by hydrogen, when 160 mM was produced in the culture medium. In co-culture with the acetogen Moorella thermoautotrophica, the hydrogen concentration decreased to a level of 4 mM due to hydrogen transfer between the two species. Consequently, the total consumption of lactose was higher for the co-culture (>80 mmol/L) than for the pure culture of C. thermolacticum (30 mmol/L). M. thermoautotrophica ...

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Heterofermentation ofClostridium thermolacticum 63  ___________________________________________________________________________
4 HETEROFERMENTATION OFCLOSTRIDIUM
THERMOLACTICUM ITS FREE-CELL CO-CULTURE WITH AND
MOORELLA THERMOAUTOTROPHICA 
4.1 Summary
The metabolites of lactose fermentation by a pure culture ofClostridium thermolacticum were lactate, acetate, ethanol, carbon dioxide and hydrogen at thermophilic temperature (60°C) and at pHs between 5.8 and 8.5. However, there was a metabolic shift from heterolactic to homolactic pathway during the fermentation, depending on the growth and pH conditions. Production of acetate and ethanol were growth associated, with an acetate to ethanol ratio of 1:1, carbon dioxide and hydrogen were formed throughout the fermentation, while lactate was produced only at the end of the exponential growth phase and during the stationary phase at a lower pH with a yield close to the maximum of 4 mol per mol lactose.C. thermolacticumwas strongly inhibited by hydrogen, when 160 mM was produced in the culture medium. In co-culture with the acetogenMoorella thermoautotrophica,the hydrogen concentration decreased to a level of 4 mM due to hydrogen transfer between the two species. Consequently, the total consumption of lactose was higher for the co-culture (>80 mmol/L) than for the pure culture ofC. thermolacticum(30 mmol/L).M. thermoautotrophica assimilated both hydrogen and lactate. Therefore, homoacetic fermentation of lactose was observed in the co-culture at pH 6.40, with an acetate to ethanol ratio of 8:1.  
Heterofermentation of 64Clostridium thermolacticum ___________________________________________________________________________  
4.2 Introduction
Acetic acid is an important organic chemical which is actually produced exclusively by petrochemical routes (Ghose and Bhadra, 1985). One important industrial use of acetic acid is to produce acetate deicers, including calcium magnesium acetate (CMA) as a noncorrosive road deicer (Chollar, 1984) and potassium acetate or sodium acetate as airport runway deicers.  Milk or whey permeates have been extensively studied as a fermentation substrate for producing lactic and propionic acids (see Chapter 2). However, its use for anaerobic acetic acid fermentation has been limited because no acetogen can directly ferment lactose to acetate. In the previous chapter, the potential feasibility of producing acetate from milk permeate fermentation using a co-culture consisting of heterofermentative and homoacetic bacteria has been studied in free-cell batch cultures without pH control. The lactose was converted to lactate byClostridium thermolacticum and then to acetate byM. thermoautotrophicawith an overall acetic acid yield of 80%. However, 13% of the fermented lactose was converted to ethanol by the heterofermentative bacterium. Actually, lactate is not produced by thermophilic bacterial fermentations in any food-processing industry. In the dairy industry, the importance of thermotolerant lactic acid-producing bacteria as starters is described in detail by Auclair and Accolas (1983). Some of these thermotolerant organisms areLactobacillus bulgaricus 40°C), (ToptBifidobacterium thermophilum, L. lactis, L. helveticusandStreptococcus thermophilus(Topt 40 to 45°C). Their use in yogurt production and their nutritional requirements have been studied by Akpemado and Bracquart (1983), and Turner et al. (1983). But the thermophilic lactic acid starters are either uncharacterized mixed cultures (artisanal starters, macerated stomach in whey) or defined mixed cultures of the above pure cultures containing various thermotolerant lactobacilli. The thermophilic bacteria are presently not yet used at large scale in food-processing or beverage-producing industry. One reason is that the renewed interest for their application is relatively recent.  Thus, among thermophilic bacteria,Clostridium thermolacticum is a known species to produce mainly lactic acid as fermentation product (Le Ruyet et al., 1985). Limited data are available on heterofermentative thermophilic clostridia and their metabolic pathways, although many species of this genus are well known as useful agents of anaerobic bioconversions. Waste treatment is one of the main areas in which anaerobic thermophilic microorganisms are being used at large scale in technical processes (Mackie and Bryant, 1981; Varel et al., 1977 and 1980). A major emphasis has been given to interspecies hydrogen transfer as the most critical reaction between acid- and alcohol-utilizers and hydrogen-utilizing methanogens and homoacetogens. The anaerobic thermophilic waste degradation is a complex process and such a microbial community is large and difficult to characterize due to the number of species and metabolic interactions involved. Also, the interactions between thermophilic heterofermentative bacteria with hydrogen-utilizing homoacetate micro-organisms has not been widely studied. Le Ruyet et al. (1984), and Kova and Nozhevnikova (1989) reported the use of a binary culture of the cellulolyticClostridium thermocellum with the acetogenAcetogenium kivui,and with the acetogenM thermoautotrophica, repectively. It was shown that in the presence of one of these two homoacetogenic bacteria, acetate is the main fermentation product. Ethanol, lactic acid, hydrogen, and carbon dioxide are present in negligible concentrations. In this binary culture the amount of broken down cellulose and the rate of its hydrolysis is higher than in a mono-culture.  
Heterofermentation ofClostridium thermolacticum 65 ___________________________________________________________________________  
The aim of this work was first to study the lactose fermentation pathway of the heterofermentative bacteriumC. thermolacticum in mono-culture in order to control its lactic acid production and to limit ethanol and gas production. Then, the free-cell co-culture fermentation ofC. thermolacticumwith the acetogenM. thermoautotrophica was studied in order to optimize acetic acid production using lactate as an intermediary product. The interactions and effects of the presence ofM. thermoautotrophica on the breakdown of lactose, the threshold concentrations and ratios of the fermentation products were finally investigated in this work and reported in this chapter.
Heterofermentation of 66Clostridium thermolacticum  ___________________________________________________________________________
4.3 Materials and methods
4.3.1 Microorganisms The heterofermentative bacteriumC. thermolacticum 2910 and the acetogenic DSM bacteriumM. thermoautotrophicawere used in this study. The stock cultures wereDSM 7417 incubated in tubes at 58°C without shaking in a synthetic medium containing either lactose or lactate as the carbon source, then stored in the refrigerator and transferred once a month to maintain good viability. The purity of the culture was routinely checked under the microscope.  4.3.2 Culture medium Unless otherwise noted, the medium contained (per liter): (NH4)2SO4, 0.5 g; NH4Cl, 0.5 g; KH2PO4, 0.33 g; K2HPO4, 0.45 g; KHCO3, 1 g; resazurin, 1mg; yeast extract, 2 g; trypticase, 2 g. A vitamin solution and a trace mineral solution were each added at 10 and 5 ml, respectively, per liter of medium. Milk permeate or lactose was also included as the carbon source in the medium for growing both the mono-culture ofClostridium thermolacticumand the co-culture withM. thermoautotrophica.Lactate was used as a carbon source for growing the mono-culture ofM. thermoautotrophica.   The mineral solution contained (per liter): nitrilotriacetic acid, 1.5 g; MgSO4, 3 g; MnSO4×H2O, 0.5 g; NaCl, 1 g; FeSO4×7H2O, 0.1 g; Co(NO3)2×6H2O, 0.1 g; CaCl2×2H2O, 0.1 g; ZnCl2, 0.1 g; CuSO4×5H2O, 0.01 g; AlK(SO4)2×12H2O, 0.01 g; H3BO3, 0.01 g; Na2MoO4×2H2O, 0.01 g; NiCl2×6H2O, 0.05 g; Na2SeO3, 0.37 mg; Na2WO4×2H2O, 0.01 g. The vitamin solution contained (mg per liter): biotin, 2; folic acid, 2; pyridoxin-HCl, 10; thiamine-HCl∙2H2O, 5; riboflavin, 5; nicotinic acid, 5; D-Ca-pantothenate, 5; vitamin B12, 0.1; p-aminobenzoic acid, 5; lipoic acid, 5.  The basal medium was boiled for 4 min, flushed with 80% N2-20% CO2gas to remove oxygen, then 0.45 g cysteine-HCl was added to reduce the medium. After having dispensed into serum tubes (12 ml each filled with 10 ml of medium) or serum bottle ( 1-L filled with 250 ml of medium) in the anaerobic chamber, the medium was autoclaved at 121°C for 35 min. The medium pH was about 7.3.  When 5-l fermentors were used, the complete bioreactor containing 3-L of the basal medium was autoclaved at 121 °C for 35 min. The sterilized medium was then flushed with filter-sterilized CO2-N2 gas to remove oxygen. Then, a separate lactose or milk permeate solution and a cysteine-HCl solution (to a final concentration of 0.5 g/L) were added aseptically through a sterile filter (pore size, 0.2mm) by the inoculation port. The medium was then immediately water-cooled at 58°C, and the pH adjusted to 6.4 or 7.0 with 5 mmol/L NaOH solution.  The milk permeate was prepared from a concentrated sweet milk permeate containing 200 g/L lactose (Cremo, Fribourg, Switzerland), which was sterilized by ultrafiltration (UFP-10-c-ss column, MM cutoff 10 000, A/G Technology, USA) and stored in a 250-liter vat at 10°C under CO2atmosphere.
Heterofermentation of 67Clostridium thermolacticum  ___________________________________________________________________________ 4.3.3 Batch culture fermentations Batch fermentations ofC. thermolacticum uncontrolled pH were performed in under serum tubes conditions to evaluate effects of pH, lactate, acetate and ethanol on cell growth. The batch fermentation ofC. thermolacticumgrown on glucose without controlled pH was performed in a serum bottle in order to analyse the gas composition.  Batch fermentations ofM. thermoautotrophica performed in serum tubes to were evaluate the pH effect on cell growth.  Metabolic pathway was also studied under controlled constant pH conditions with 5-liter fermentors (Meredos, Switzerland) for the mono- and co-culture. The fermentor broth was automatically titrated with 5 M NaOH to maintain a desired pH. A total of 3 liters of medium was used for each batch fermentation, and 60 ml of active culture was inoculated into the medium. At proper time intervals (depending on the fermentation rate), liquid samples (3 ml) were withdrawn from the sampling port for OD reading and HPLC analysis.  4.3.4 Analytical techniques Organic compoundsLactose, glucose, galactose, lactate, acetate and ethanol.  were identified and quantified by high-performance liquid chromatography (HPLC). The HPLC system consisted of a pump (Varian 9012), an automatic injector (Varian 9100), and a differential refractometer at 45°C (ERC-7515, Erma CR-INC). Samples were deproteinated, centrifuged and finally filtered through a 0.2-µm membrane filter to remove bacterial cells. Then, 20 µL of filtrate were injected onto an organic acid column (Interaction ORH-801) at 60°C. Elution was done by 0.005 M sulfuric acid at a flow rate of 0.8 mL/min. Calibration curves for standards of each compound were done. The accuracy of this analysis was higher than 95 % with daily control of the calibration.  H2 and CO2 were determined using a type F20H Perkin-Elmer gas chromatograph with thermal conductivity detector and 2-m glass column containing 5A molecular sieve (E. Merck, AG, Switzerland). To analyze gases solubilized in the culture fluid, 1-mL sample was transferred to a 4.5-mL stoppered serum bottle containing 1 mL concentrated sulfuric acid to liberate CO2. After bottles had been shaken to equilibrate the gas phase with the acidified sample, a 200-mL sample of the gas phase was analyzed as described above. The total pressure inside serum bottles was measured with a digital pressure meter (Galaxy). The amount of gas (H2 CO or2) produced per unit volume of the liquid medium (mol per L) was then calculated from the gas composition (%), total pressure (Pa) and gas volume (m3) inside the bottle, and temperature (K) as follow:  
l ress[ ]×Volume  PercentageH2or CO2  ×oTat31.8Jpmolu1rePK1aTemperagtasurem [3K]×loV1emumiuedm[L] ×-×-×  Cell densitywas monitored by measuring the OD at 650 nm in a spectrophotometer (Hitachi, U-2000). A direct measurement of OD in the growth tube was made for cultures grown in the tubes. Otherwise, OD was measured in 1.5 ml acrylic cuvettes. Samples were diluted with water if OD readings were greater than 0.5. One unit of OD was found to be equivalent to 1.16 g (dry weight) ofC. thermolacticumcells per liter and 0.15 g forM. thermoautotrophica.
Heterofermentation ofClostridium thermolacticum 68  ___________________________________________________________________________
4.4 Results
4.4.1 Fermentation characteristics ofC. thermolacticum  pH range for growth.It is necessary to find an appropriate pH for both the mono-culture ofC. thermolacticum the co-cultured fermentation with andM. thermoautotrophica. pH Thus, range for growth was studied first. By using data from the early exponential phase of growth, the specific growth rates at various pH values were determined, and the effect of the pH on the specific growth rate is shown in Figure 4.1 (b). Apparently, this bacterium has an optimal pH for growth at around pH 7.3 with a specific growth rate of~0.25 h-1in the presence of 10 g/L of lactose. The fermentation rate was relatively insensitive to pH changes between 6.5 and 8.2 but decreased dramatically at a pH below 6.5 or above 8.2. No growth was found for cells cultivated at a pH below 5.9 or above 8.7 for a period of two weeks, indicating a growth pH range between 5.9 and 8.7. However, for uncontrolled pH experiments, cultures could continue to grow until the medium pH decreased to 5.0 (cf Chapter 3, Fig. 3.1). Thus, this pH value is significatively lower then the observed minimum initial medium pH for cell growth. Either the active cells could tolerate a lower pH than did the dormant cells (because actively grown cells may establish a pH gradient by an ATPase-driven extrusion of protons from the cells), or some internal acids were released to the medium after cells had dropped growth, causing the pH to continue to drop below 5.9 (cf Chapter 3 about lactic acid production during the exponential phase of growth).   The pH range for growth ofM. thermoautotrophicaon lactate was between 5.5 and 7.7, with an optimal pH at around 6.5 (see Chater 3 and Figure 4.1 (a)). Thus, the optimum pH for the mixed culture fermentation should be around 6.5.
Heterofermentation ofClostridium thermolacticum 69 ___________________________________________________________________________   
 
0.06 0.05 0.04 0.03 0.02 0.01 0 4.5 a.
0.25
0.2
0.15
0.1
0.05
5 5.5 6 6.5 7 7.5 8 8.5
9
0 b.5 5.5 6 6.5 7 7.5 8 8.5 9 pH Figure 4.1. Effect of pH on the growth at 58°C ofMoorella thermoautotrophica(a) andClostridium thermolacticum(b).
Heterofermentation ofClostridium thermolacticum 70 ___________________________________________________________________________  Effects of acetic acid, lactic acid and ethanol. Fermentation products were found to inhibit strongly the heterolactic fermentation (see Figures 4.2 and 4.3). The specific growth rate ofC. thermolacticum was reduced to zero in the presence of 25 g/L of acetic acid (420 mM), or 20 g/L of lactic acid (220 mM), or 10 g/L of ethanol (250 mM) at pH 7.3 (no growth were obtained after 1 week of incubation). Thus, this bacterium was more strongly inhibited by ethanol and lactic acid than by acetic acid. The effect of acetic acid was found to be stronger at a lower medium pH (complete inhibition at 18 g/L of acetate at pH 6.40), indicating that the undissociated acid may be more inhibitory than the dissociated acid. As shown on Figure 4.3, the specific growth rate seemed not to be affected by an increasing ethanol concentration until 10 g/L but the lag phase durations increased.   
       
0.25
0.2 0.15
0.1 0.05
0 0
Lactate
Acetate
5 10 15 20 Fermentation product concentration (g/L)
 Figure 4.2. Effects of acetic and lactic acids on specific growth rate of Clostridium thermolacticumat pH 7.3, 58°C and 10 g/L of lactose.
25
Heterofermentation of 71Clostridium thermolacticum  ___________________________________________________________________________  
0.7
0.6 0.5 3.92 g/L 0 g/L 0.4e thanol 1.23 g/L 0.3 5.24 g/L 0.2 0.1
6.69 g/L
8.12 g/L
9.58 g/L
0 0 10 20 30 40 50 60 70 80 Time (h)
 Figure 4.3. Effects of ethanol concentration on growth ofClostridium thermolacticumat pH 7.3, 58°C and 10 g/L of lactose.   Heterofermentation of C. thermolacticum on glucose. Figure 4.4 (b) shows typical kinetics of fermentation ofC. thermolacticum grown on glucose. Products from this fermentation included lactate, acetate, ethanol, carbon dioxide and hydrogen. In this batch culture, cell growth stopped when only 26.62 mmol/L of glucose had been consumed, probably because of an effect of pH on cell growth. Actually, pH was not controlled during fermentation, and the medium pH dropped from an initial value of 7.4 to 5.0 when cell growth stopped (see Figure 4.4 (a)). During the exponential phase of growth, acetate, ethanol, CO2and H2were produced. However, neither ethanol nor acetate were produced once cells reached the stationary phase, indicating that their production was growth-associated, while CO2 and H2 production continued until the end of the fermentation (see Figure 4.4 (c)). On the other hand, lactate, not present during the exponential phase of growth, was produced only during the stationary phase, with a yield of 1.5 mole formed per mole of glucose consumed. Based on the carbon balance calculation, about 94 % of the glucose fermented was converted into the various metabolites and 6 % was incorporated into cell biomass. The final product molar ratio in this fermentation was: lactate (1.03), acetate (0.27), ethanol (0.36), CO2(1.29), H2(1.05).  
Heterofermentation of 72Clostridium thermolacticum ___________________________________________________________________________  
   
8 7.5 7 6.5 6 5.5 5 a.0 20 40 60 70 60Cell 50 Glucose 40 CO2 30H2 20Lactate Ethanol 10 Acetate 0 b. 20 30 40 50 60 700 10 40 35 CO2Cell 30 25H2 20Lactate 15 Ethanol 10 5Acetate 0 c.35 40 45 50 55 60 65 Glucose concentration (mM)
80
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 80 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 70
Figure 4.4. Batch culture ofClostridium thermolacticum on glucose grown at 58°C without controlled pH.
Heterofermentation ofClostridium thermolacticum 73 ___________________________________________________________________________  
4.4.2 Mono-culture ofC. thermolacticumunder controlled pH conditions Batch fermentations at controlled constant pHs between 7.40 and 6.20 were also studied in bioreactor with pure lactose or milk permeate as substrate (see the summary of the results in Table 4.1). Results from these experiments were consistent with those of previous uncontrolled pH experiments in test tubes or in serum bottles (see Chapter 3).  Fermentation of C. thermolacticum on lactose.Figure 4.5 shows typical fermentation kinetics ofC. thermolacticumgrown on lactose at pH 7.40 and 6.40. At a pH of 7.40 (Figure 4.5 (a)), fermentation products included acetate, ethanol, CO2 H and2. In such batch culture, cell growth stopped when only 30 mmol/L of lactose had been consumed, and 26 and 32 mmol/L of acetate and ethanol, respectively, had been produced during the exponential growth phase. Only a small amount of lactate was formed at pH 7.40 at the end of the fermentation with a yield of 1.6 mol/mol. Hydrolysis of lactose continued even after the fermentation had stopped. According to the cell dry weight calculation, about 91 % of the lactose was converted into the various metabolites and 9 % was incorporated into cell biomass. Based on the carbon balance, the final product molar ratio in this fermentation was: lactate (0.13), acetate (0.83), ethanol (1.06) and CO2(5.26).  At pH 6.40 (Figure 4.5 (b)), the fermentation stopped when 66 mmol/L of lactose had been consumed, which was approximately twice the amount of lactose consumption in the previous batch culture at pH 7.40. But similar to the previous batch, the fermentation stopped after 34 and 45 mmol/L of acetate and ethanol produced during the exponential phase. However, while little lactate was formed at pH 7.40, at pH 6.40 lactate formation was delayed in the growth phase and continued to the stationary phase with a maximal yield of 4 moles of lactate produced per mole of lactose consumed.  It seems that the fermentation started to be inhibited after 90 h, as seen by the accumulation of glucose and galactose in the medium broth. Then, since sugars were still slowly consumed, more lactose was added in the medium from the inoculation port. Surprisingly, the bacterial cells restarted to grow and lactate was further produced with a maximal yield of 4 mol per mol of lactose consumed, without acetate and ethanol production. Then, the sugar consumption stopped completely with a final lactate concentration of 127 mmol/L. The lactate, acetate and ethanol concentrations were lower than the inhibitory concentrations determined previously, suggesting that hydrogen was the inhibitory factor of the fermentation. Consequently, the hydrogen produced during the first part of the fermentation was probably stripped during the medium purge when lactose was added, removing the inhibition and allowing the cells to grow during the second part of the fermentation. Then, hydrogen was probably produced and coupled with the lactate production (as seen with the glucose fermentation), inhibiting the fermentation.