Anaerobic thermophilic fermentation for acetic acid production from milk permeate

Journal of Biotechnology 76 (2000) 83–92 Anaerobic thermophilic fermentation for acetic acid production from milk permeate Myle`ne Talabardon *, Jean-Paul Schwitzgue´bel, Paul Pe´ringer Laboratory for En6ironmental Biotechnology, Swiss Federal Institute of Technology of Lausanne ( EPFL ) , Ecublens, CH- 1015 Lausanne, Switzerland Received 27 January 1999; received in revised form 26 July 1999; accepted 30 July 1999 Abstract Fermentation of milk permeate to produce acetic acid under anaerobic thermophilic conditions ( 60°C) was studied. Although none of the known thermophilic acetogenic bacteria can ferment lactose, it has been found that one strain can use galactose and two strains can use lactate. Moorella thermoautotrophica DSM 7417 and M. ther- moacetica DSM 2955 were able to convert lactate to acetate at thermophilic temperatures with a yield of  0.93 g g −1 . Among the strains screened for their abilities to produce acetate and lactate from lactose, Clostridium thermolacticum DSM 2910 was found precisely to produce large amounts of lactate and acetate. However, it also produced significant amounts of ethanol, CO 2 and H 2 . The lactate yield was affected by cell growth. During the exponential phase, acetate, ethanol, CO 2 and H 2 were the main products of fermentation with an equimolar acetate/ethanol ratio, whereas during the stationary phase, only lactic acid was produced with a yield of 4 mol per mol lactose, thus reaching the maximal theoretical value. When this bacterium was co-cultured with M. thermoau- totrophica, lactose was first converted mainly to lactic acid, then to acetic acid, with a zero residual lactic acid concentration and an overall yield of acetate around 80%. Under such conditions, only 13% of the fermented lactose was converted to ethanol by C. thermolacticum. © 2000 Elsevier Science B.V. All rights reserved. Keywords : Screening; Clostridium; Moorella; Lactose; Heterofermentation; Acetogens www.elsevier.com/locate/jbiotec 1. Introduction In Switzerland, cheese industry produces large amounts of lactose in the form of milk permeate or whey permeate. Ultrafiltration is frequently used for concentrating milk in several large cheese producing plants (e.g. Feta cheese) as well as in manufacturing special milk products. This cheese- making technology produces, instead of whey, a deproteinated permeate which needs further pro- cessing. The permeate contains about 5% lactose, 1% salts, and 0.1–0.8% lactic acid; it is practically free of N-compounds and thus not comparable with whey which contains up to 0.8% protein (Ka¨ppeli et al., 1981). Because of its lack of * Corresponding author. Present address: Department of Chemical Engineering, Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA. Fax: + 1-614-292-3769. E-mail address : mylene.talabardon@epfl.ch (M. Talabar- don) 0168-1656/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S0168-1656(99)00180-7 M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83 – 92 84 protein, it is unsuitable for animal or human feeding. It has a high chemical oxygen demand of 57 to 65 g l −1 or even higher, depending on the cheese manufacturing process and is a major dis- posal problem of overloading to sewage treatment plants. This lactose source, being directly fer- mentable by many bacteria and presently being a negative value waste stream on account of the expensive wastewater treatment before discharge, could serve as an excellent feedstock for the pro- duction of acetic acid. Anaerobic acetogenesis conserves all the carbon of glucose in the product acetic acid, thus increas- ing overall yield per glucose molecule by 50% over the aerobic vinegar process (Busche, 1991). Acetic acid production from glucose by Moorella ther- moacetica under thermophilic conditions appears to be feasible (Shah and Cheryan, 1995). With 45 gl −1 of glucose in the feed of a fed-batch bioreac- tor and a two-stage CSTR, the productivity and the concentration of acetic acid are 1.12 g l −1 ·h −1 and 38 g l −1 , respectively. Although most thermophilic acetogens can convert glucose to acetate with a product yield as high as  90% (Wiegel, 1994), there is no known thermophilic acetogen able to produce acetate from lactose directly. Bream (1988) has isolated a mutant of M. thermoacetica able to grow on lactate as the only source of carbon and energy, whereas the parent strain consumes lactate only in the pres- ence of a second fermentable substrate. With the mutant strain, it is possible to produce acetate from lactose through lactate as an intermediary fermentation step. Anaerobic fermentations to produce acetic acid from whey lactose have been studied under mesophilic conditions. Tang et al. (1988) have reported the use of Lactobacillus lactis and Clostridium formicoaceticum on sweet whey per- meate. The former is a homolactic bacterium, which converts lactose to lactate, and the latter can produce acetate from lactate. A new fermen- tation process has recently been developed by Huang and Yang (1998) using this co-culture immobilized in a fibrous-bed bioreactor. Under fed-batch fermentation conditions, a final acetate concentration of 75 g l −1 and an overall produc- tivity of 1.23 g l −1 ·h −1 were obtained. However, a thermophilic fermentation process could be more interesting, since it has generally a higher production rate, should be more resistant to con- tamination and more convenient to maintain anaerobic conditions required for acetogens. In this work, several potential ways for lactose fermentation to acetic acid under anaerobic ther- mophilic conditions ( 60°C) were studied. Dif- ferent heterofermentative and acetogenic bacteria were evaluated for their potential use to produce acetate, and a co-culture of two bacteria, Clostrid- ium thermolacticum and M. thermoautotrophica, was found to give high acetate yield from lactose. 2. Materials and methods 2 . 1 . Microorganisms The heterofermentative and acetogenic bacteria used in this study are listed in Tables 1 and 2, respectively. The freeze-dried strains were first hydrated in a minimal volume of fresh culture medium in an anaerobic chamber, and then trans- ferred anaerobically in serum bottles. Bacteria in spore phase (or in the exponential growth phase for non-sporulating species) were stored at 4°C and used as stock cultures. The purity of cultures was routinely checked under microscope (phase contrast). The heterofermentative bacterium C. thermo- lacticum DSM 2910 and the acetogenic bacterium M. thermoautotrophica DSM 7417, used in this study, were isolated from a mesophilic digester fed with Lemna mina (France) by Le Ruyet et al. (1984), and from a pectin-limited culture of Clostridium thermosaccharolyticum by van Rissjel et al. (1992), respectively. 2 . 2 . Culture media Each bacterial strain was cultivated in the medium as specified in the DSM or ATCC cata- logues. Unless otherwise noted, the medium used in the fermentation study was prepared as follows. The basal medium (see medium 326 in the DSM catalogue) contained (per liter in deionized water): K 2 HPO 4 , 0.348 g; KH 2 PO 4 , 0.227 g; NH 4 Cl, 2.5 M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83 – 92 85 Table 1 Summary of screening results for various thermophilic heterofermentative bacteria grown on milk permeate Thermoanaero- Thermoanaerobacter ethanolicus Thermoanaero-Clostridium thermolacticum Thermoanaerobacter thermohy- Thermoanaero-Species drosulfuricus bacterium ther-bacter finniibacter brockii ssp mosaccha-brockii rolyticum DSM 3389 T Strain DSM 2247 T DSM 2910 T DSM 567 T DSM 571 T DSM 2911 T DSM 1457 T DSM 2246 T DSM 2355 T Schmid et al.,Le Ruyet et al., 1984, 1985 Wiegel et al., 1979; Hollaus andFrom the refer- Wiegel and Ljungdahl, 1981; Wiegel, 1992Zeikus et al., 1986Zeikus et al., 1980 Klaushofer, 19731979ences Temperature 50–70 (65) 35–85 (65–70) 37–78 (69) 40–75 (65) 37–78 (67–69) 35–67 (55)50–70 (60–65) range (°C) (optimal tem- perature) pH range for 5.5–9.5 (7.5)6.0–7.8 (7.0–7.2) 4.4–9.8 (5.8–8.5) (6.5–6.8) 5.5–9.2 (6.9–7.5) 7.0–8.56.0–7.8 (7.2–7.4) growth (opti- mal pH for growth) From the present study 25.57 31.42 29.74 20.02 42.6515.02 35.38Lactose fer- 56.5013.04 mented (mmol l −1 ) Temperature 65 65 65 65 65 606560 65 (°C) Initial pH 7.417.68 7.52 7.77 7.707.42 7.40 7.57 7.28 4.76 4.70 4.90 4.824.805.90 4.71Final pH 4.765.84 Product yield (mol mol −1 ) 2.38 0.69 0.79 01.242.45Lactate 0.961.142.02 0.230.73 0.26 0.20 0.58 0.291.00 0.19 0.30Acetate 0.98 2.87 1.26 1.38Ethanol 0.73 1.00 1.76 1.63 2.06 2.33 3.5 4.80 8.414.71.46 a CO 2 4.563.942.00 a 0.241.46 a 0.59 0.34 3.67 7.442.00 a 0.53 0.27H 2 Other fermenta- ––––––––+ tion products detected by HPLC but not identified 0.88 1.31 0.610.40 1.87Biomass (by dry 0.97 0.640.43 0.98 weight in g l −1 ) 98.00 97.5 97.58 90.42 97.98% carbon 97.00 97.00 93.83 94.23 recovery b 0.59 0.60 2.43 0.24 0.632.02 03.36 0.65Ratio mol lac- tate/mol ethanol a Calculated by carbon balance. b The percentage of carbon recovery was calculated as the ratio: total carbon present in all fermentation products/total carbon in fermented carbon sources. M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83 – 92 86 g; NaCl, 2.25 g; FeSO 4 ·7H 2 O, 0.002 g; yeast extract (Difco), 2 g; resazurin, 0.001 g; trace ele- ment solution, 1 ml. The trace element solution SL-10 (see medium 320 described in the DSM catalogue) contained (per liter in 0.077 mmol l −1 HCl): FeCl 2 ·4H 2 O, 1.5 g; ZnCl 2 ,70mg; MnCl 2 ·4H 2 O, 100 mg; H 3 BO 4 , 6 mg; CoCl 2 ·6H 2 O, 190 mg; CuCl 2 ·2H 2 O, 2 mg; NiCl 2 ·6H 2 O, 24 mg; Na 2 MoO 4 ·2H 2 O, 36 mg. Each serum bottle (1 l) containing 250 ml of the basal medium was flushed with 20% CO 2 /80% N 2 gas to remove oxygen, then autoclaved at 121°C for 20 min. After autoclaving, additional nutrients contained in a concentrated solution were added to the basal medium, by passing through a mi- crofilter (0.45 mm pore size), to the following final concentrations (per liter of basal medium): 0.5 g MgSO 4 ·7H 2 O, 0.25 g CaCl 2 ·2H 2 O, 4.5 g KHCO 3 , 0.3 g cysteine-HCl · H 2 O, 0.3 g Na 2 S·9H 2 O, 10 ml vitamin solution (see below), and 20 g of a carbon source selected from lactose, milk permeate, glucose, galactose, or DL-sodium lactate. The milk permeate was prepared from a frozen, concentrated sweet milk permeate contain- ing 200 g l −1 lactose (Cremo, Fribourg, Switzer- land), which was sterilized by ultrafiltration (UFP-10-c-ss column, MM cutoff 10 000, A/G Technology, USA) and stored in a 250 l vat at 10°C under CO 2 atmosphere. The vitamin solu- tion (see medium 141 in the DSM catalogue) contained (in mg l −1 ): biotin, 2; folic acid, 2; pyridoxin-HCl, 10; thiamine-HCl · 2H 2 O, 5; ri- boflavin, 5; nicotinic acid, 5; D-Ca-pantothenate, 5; vitamin B 12 , 0.1; p-aminobenzoic acid, 5; lipoic acid, 5. The pH of the medium was adjusted to the desired value with a filter-sterilized NaOH or HCl solution. It is noted that the spores of thermophiles are heat resistant and all medium bottles used in this study were not mixed for different strains, which allowed us to use the less stringent sterilisation conditions without the risk of cross contamina- tion. However, for the stock cultures, media con- taining all components were autoclaved for 45 min at 121°C to ensure complete sterilisation. Any medium components that were heat labile were sterilised with a sterile 0.2 mm filter. 2 . 3 . Batch culture fermentations All batch fermentation studies were performed in 1 l screw-capped serum bottles, with 250 ml of medium, and fitted with gas-impermeable black butyl rubber septa under anaerobic and non-con- trolled pH conditions, in a constant temperature incubator (58°C, agitation speed: 100 rpm). Fif- teen milliliters of a spore or cell (in the exponen- tial growth phase) suspension were added as inoculum to each serum bottle. For the spore inoculum, a heat treatment (5 min at 105°C) was used to kill vegetative cells and to activate spores. Liquid samples (5 ml each) were taken with sterile Table 2 Screening results of various thermophilic acetogens cultivated in media containing galactose or lactate as sole carbon source Species Strain Acetate yield from galactose (mol/mol) Acetate yield from lactate (mol/mol) Calorimator fer6idus DSM 5463 T – a – DSM 2030 T Acetogenium ki6ui –– Acetomicrobium fla6idum DSM 20664 T –– 1.40–1.46Moorella thermoacetica –DSM 2955 T DSM 521 T –– DSM 6867 T –– DSM 39073 T –– ATCC 34490 T –– ATCC 39289 T –– Moorella thermoau-–DSM 7417 T 1.38–1.46 totrophica 2.0–2.5DSM 1974 T – a No growth is indicated by –. M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83 – 92 87 syringes throughout the batch fermentation for optical density (OD) reading, pH measurement, and HPLC analysis. 2 . 4 . Analytical techniques Lactose, glucose, galactose, lactate, acetate and ethanol were identified and quantified by high-per- formance liquid chromatography (HPLC). The HPLC system consisted of a pump (Varian 9012), an automatic injector (Varian 9100), and a differ- ential refractometer at 45°C (ERC-7515, Erma CR-INC). Samples were deproteinated, centrifuged and finally filtered through a 0.2 mm membrane filter to remove bacterial cells. Then, 20 ml of filtrate were injected onto an organic acid column (Inter- action ORH-801) at 60°C. Elution was done by 0.005 mmol l −1 sulfuric acid at a flow rate of 0.8 ml min −1 . Calibration curves for standards of each compound were done. The accuracy of this analysis was higher than 95% with daily control of the calibration. H 2 and CO 2 were determined using a type F20H Perkin-Elmer gas chromatograph with thermal conductivity detector and 2-m glass column con- taining 5A molecular sieve (E. Merck, AG, Switzer- land). To analyze the gases solubilised in the culture fluid, 1 ml sample was transferred to a 4.5 ml stoppered serum bottle containing 1 ml concen- trated sulfuric acid to liberate CO 2 . After the bottle 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 the serum bottle was measured with a digital pressure meter (Galaxy). The amount of gas (H 2 or CO 2 ) produced per unit volume of the liquid medium (mol per liter) was then calculated from the gas composition (%), total pressure (Pa) and gas volume (m 3 ) inside the bottle, and temper- ature (K) as follows: Cell density was monitored by measuring the optical density at 650 nm (OD 650 ) in a spectropho- tometer (Hitachi, U-2000). Samples were diluted when OD was greater than 0.5. The biomass was calculated by dry weight. A calibration curve, OD versus dry weight, was done for each strain. 3. Results 3 . 1 . Screening Two major groups of bacteria, including hetero- fermentative and acetogenic bacteria, that might be involved in the acetic acid fermentation were screened (Tables 1 and 2). All anaerobic acetogens, carrying out a homoacetogenic fermentation, can utilize glucose and CO 2 and H 2 to produce acetate, but none can grow on lactose. However, lactose can be readily hydrolyzed to glucose and galactose by many fermentative bacteria or the b-galactosidase enzyme. Thus, 12 known thermophilic homoaceto- gens were screened for their abilities to ferment galactose. Table 2 shows that only M. thermoau- totrophica DSM 1974 was able to use galactose when this substrate was present as the only source of carbon and energy, producing 2.5 mol acetic acid per mol of galactose consumed. However, this bacterium fermented only glucose when both glu- cose and galactose were present in the medium. Consequently, this bacterium was not suitable to produce acetic acid from hydrolyzed milk perme- ate. Among the 12 acetogens screened, two strains produce acetate from lactate. M thermoau- totrophica DSM 7417 and M. thermoacetica DSM 2955 produced  1.4 mol acetic acid per mol lactic acid consumed (0.93 g g −1 ). The growth and degradation rates were very similar for both strains: the pH range for cell growth was between 5.0 and 7.8, with an optimal pH at  6.5. The optimal temperature was reported to be at 58°C, although they can grow at a temperature as high as 68°C (Wiegel, 1992). Meanwhile there are many mesophilic and thermo-tolerant homolactic bacteria that can con- vert lactose to lactate, such as Lactobacillus bulgari- cus, Bifidobacterium thermophilum, L. lactis and L. Percentage H 2 or CO 2 · total pressure [Pa] · volume gas [m 3 ] 8.31[J mol -1 K -1 ]·temperature [K] · 1 volume medium [L] M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83 – 92 88 Fig. 1. Batch culture of Clostridium thermolacticum DSM 2910 grown on lactose, at 58°C, initial pH 7.32, and 100 rpm agitation speed (with 6% inoculation in exponential phase). ethanol, CO 2 and H 2 . As can be seen in Table 1, Clostridium thermolacticum DSM 2910 appears to be an appropriate strain for lactate production on account of its high lactate yield (2.45 mol per mol lactose fermented) and high lactate/ethanol ratio (3.36 mol mol −1 ). This bacterium had an optimal growth temperature at 60°C and growth pH range between 6.0 and 7.8. 3 . 2 . Fermentation of C. thermolacticum on lactose and milk permeate To further evaluate the fermentation way to produce acetate from lactose via lactate, H 2 and CO 2 using heterofermentative and acetogenic bac- teria, detailed fermentation kinetics were studied and the results are reported here. Fig. 1 shows typical kinetics of fermentation of C. thermolacticum grown on lactose. Products from this fermentation included lactate, acetate, ethanol, CO 2 and H 2 . In such batch cultures, cell growth stopped when only  18 mmol l −1 of lactose 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 7.32 to  5.9 when cell growth stopped. During the exponential phase of growth, acetate, ethanol, CO 2 and H 2 were produced, while lactate forma- tion was relatively small and was delayed. How- ever, neither ethanol nor acetate was produced once cells reached the stationary phase, indicating that their production was growth-associated. On the other hand, more lactate was produced in the stationary phase. The drop of pH generally coin- cided with acids production. It was also obvious that lactose was hydrolyzed to glucose and galac- tose, which accumulated in the broth, when cell growth was low. Hydrolysis of lactose, continued even after the fermentation had stopped, possibly catalyzed by the b-galactosidase enzyme released during the sporulation. Based on the carbon bal- ance calculation, about 97% of the lactose fer- mented was converted into the various metabolites and only  3% was incorporated into cell biomass. The final product molar ratio in this fermentation was approximately: lactate (1), ac- etate (1), ethanol (1), CO 2 (5), H 2 (5). hel6eticus; there is only one known thermophilic homolactic bacterium, Streptococcus (Lactobacil- lus) thermophilus. However, the production of lactic acid from sugars by this bacterium at ther- mophilic temperatures (\ 50°C) is poor (Wiegel and Ljungdahl, 1986) because of its fastidious growth requirements. Thus, S. thermophilus is usually considered as unsuitable for thermophilic production of lactic acid and has only been used at mesophilic temperatures (up to 45°C) in co-cul- ture with the mesophilic L. hel6eticus (Boyaval et al., 1988). Therefore, various anaerobic ther- mophilic strains, belonging to the saccharolytic or cellulolytic group of bacteria, were screened for their abilities to produce acetic acid from lactose present in milk permeate. Results are summarized in Table 1: all were obtained from batch fermen- tation experiments without pH control or any other attempt to optimize the fermentation condi- tions. Among these strains, there was no ther- mophilic homolactic bacterium, and the main fermentation products were lactate, acetate, M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83 – 92 89 The pattern of growth and fermentation in a medium containing 25 mmol l −1 lactose from milk permeate is shown in Fig. 2. Products from this fermentation included lactate, acetate, ethanol, and CO 2 and H 2 (not shown). In this batch culture, the fermentation almost stopped when 13 mmol l −1 of lactose had been consumed. Similar to the previous fermentation with lactose as the substrate, acetate and ethanol were only produced during the exponential growth phase, and lactate formation was delayed in the growth phase but continued to the stationary phase. However, more lactate and less gases (CO 2 and H 2 ) were produced in this batch as compared to the previous one (Fig. 1). The final product molar ratio in this batch was approximately: lactate (3), acetate (1), ethanol (1), CO 2 (2), H 2 (2). Because of its content in phosphate ( 1.5gl −1 ), milk permeate has a higher buffer capacity than the basic medium used in the previous experiment. Therefore, the decrease of pH was lower and slower under such growth conditions. Fig. 3. Batch co-culture of Clostridium thermolacticum DSM 2910 and Moorella thermoautotrophica DSM 7417 grown on milk permeate at 58°C, initial pH 7.2 with 40 mM MOPS (buffer), and 100 rpm agitation speed (with 6% inoculation in spore phase for each species). Fig. 2. Batch culture of Clostridium thermolacticum DSM 2910 grown on milk permeate, at 58°C, initial pH 7.68, and 100 rpm agitation speed (with 6% inoculation in spore phase). 3 . 3 . Acetogenic fermentation of M. thermoautotrophica on lactate M. thermoautotrophica DSM 7417 homofer- mentatively converted lactate to acetate at ther- mophilic temperature (50–65°C) and at pH between 5.8 and 7.7 (not shown). Approximately 0.93 g of acetic acid was formed from each gram of lactic acid. The bacterium grew at an optimal pH of 6.35–6.85 and an optimal temperature of 58°C. This bacterium was thus chosen for use in a fermentation with a mixed culture to produce acetic acid from milk permeate. 3 . 4 . Fermentation of the co-culture of C. thermolacticum and M. thermoautotrophica on milk permeate Fig. 3 shows a typical time course of batch fermentation of lactose by the co-culture of C. thermolacticum DSM 2910 and M. thermoau- M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83 – 92 90 totrophica DSM 7417. As can be seen in this figure, acetic acid was the major product, with a yield of 4.83 mol mol −1 (0.81 g g −1 ) lactose fermented, and ethanol was only produced at the beginning of the fermentation, with a final yield of 0.77 mol mol −1 . There was no lactic acid accumulation in the fermentation broth, suggest- ing all lactate produced by the heterofermenta- tive bacterium was completely converted to acetate by the acetogen. Because the medium pH was not controlled and dropped below 6.0 in the medium, C. thermolacticum stopped its lactose consumption, as evidenced by the accumulation of glucose and galactose in the broth. However using pH-controlled fermentation should solve this problem. It was noted that even after lactose and lactate had been completely utilized, there was continued production of acetate, which was attributed to the acetogenic growth of M. ther- moautotrophica on CO 2 and H 2 . 4. Discussion 4 . 1 . Ways for acetic acid fermentation from lactose Numerous anaerobic bacteria produce acetate as one of the fermentation products. However, there is no known strain able to produce acetate as the only fermentation product directly from lactose. Thus, it is necessary to convert lactose to lactate and then to acetate using a mixed culture consisting of two different groups of thermophilic anaerobic bacteria. All the screened thermophilic bacteria also produced acetate, ethanol, CO 2 and H 2 from lactose, although lac- tate was the major fermentation product in sev- eral strains. However, these by-products, including CO 2 and H 2 , from the heterofermenta- tion can be readily converted to acetate by most acetogens. In this work, the possibility to pro- duce acetic acid from milk permeate in anaero- bic thermophilic fermentation was demonstrated with a mixed culture of C. thermolacticum and M. thermoautotrophica ; the former for lactic acid production from milk permeate, the latter for acetic acid production from lactic acid. In batch culture experiments without pH control, an ac- etate yield of 4.83 mol per mol lactose fermented was obtained. The overall acetate yield from lac- tose can be further improved by optimizing the fermentation conditions (e.g. pH and medium composition) that may affect cell growth and the fermentation pathway used in the heterofermen- tative bacterium. As shown on Figs. 1 and 2, acetate and ethanol were produced from lactose by C. ther- molacticum only during the exponential phase of growth, whereas the production of lactate oc- curred mainly in the stationary phase. Appar- ently, there was a metabolic shift from heterofermentative to homolactic pathway de- pendent on the growth phase. The production of both acetate and ethanol was growth associated, with the same yield of 1– 2 mol per mol lactose fermented in the exponential phase. For each mol of acetate produced, there would be 2 –5 mol of CO 2 and H 2 released. The production of CO 2 and H 2 also seemed to stop soon after cells entered the stationary phase, and only lactate was thus produced from lactose, with a product yield close to the theoretical maximum of 4 mol per mol lactose. It is thus concluded that the heterofermentative bacterium, C. thermolacticum, could perform homolactic acid fermentation when its growth was limited and cells were in the stationary phase. Work is underway to opti- mize the conditions to favor lactate production from lactose by this bacterium. 4 . 2 . Benefits of fermentation with co-culture In the fermentation with a co-culture, interac- tions between both bacterial species might have also helped to shift the heterofermentative path- way to favor the transient production of lactate and the accumulation of acetate, instead of ethanol, CO 2 and H 2 . The yield of acetic acid observed in the co-culture, 0.81 g g −1 , was higher than the yield obtained (0.73 g g −1 ) when lactose was sequentially converted to lactic acid, then to acetic acid in two successive batch fer- mentations. As already seen in Fig. 3, lactate served as a good intermediary product: all lac- tate produced from the first bacterium was M. Talabardon et al. / Journal of Biotechnology 76 (2000) 83 – 92 91 timely converted to acetate by the acetogen. It should be noted that high concentrations of lac- tate could inhibit both C. thermolacticum and M. thermoautotrophica. This double (product and substrate) inhibition problem was avoided if both bacteria were cultivated in the same vessel. Thus, it should be advantageous to use a one- stage co-culture for acetate production from milk permeate. It is clear that more than 95% of lactose could be converted to acetic acid in this co-cul- ture if the ethanol produced could also be con- verted to acetic acid or if the heterofermentation could be shifted to homolactic acid fermenta- tion. To also convert ethanol to acetic acid would require a tri-culture to complete the fer- mentation. It is known that Moorella ther- moacetica ATCC 39073, although unable to couple the oxidation of ethanol to acetogenesis, is competent in ethanol-dependent growth when ethanol oxidation is coupled to the reduction of dimethylsulfoxide or thiosulfate (Beaty and Ljungdahl, 1991). This possibility, however, re- mains to be tested. It is thus better and simpler to shift the heterofermentation to homolactic fermentation by controlling the fermentation conditions and growth phases, as demonstrated in this study. Furthermore, it is possible to use immobilized cell fermentation to reduce cell growth and increase product yields (Huang and Yang, 1998). Better acetic acid production from lactose can be obtained if the co-culture of C. thermolacticum and M. thermoautotrophica are maintained in the stationary phase by immobi- lizing the cells in a fibrous-bed bioreactor (Tal- abardon, 1999). As already discussed before, when the heterofermentative bacteria were in the non-growing state or stationary phase, the het- erofermentative pathway shifted to the homolac- tic acid pathway and only lactate was produced from lactose with a nearly 100% yield. It is thus possible to produce acetate from lactose with a yield higher than 95% by using this thermophilic co-culture. 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