SCREENING OF ANAEROBIC THERMOPHILIC BACTERIA

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Screening of anaerobic thermophilic bacteria 43 ___________________________________________________________________________ 3 SCREENING OF ANAEROBIC THERMOPHILIC BACTERIA 3.1 Summary 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 Moorella thermoacetica DSM 2955 were able to convert lactate to acetate at thermophilic temperatures with a yield of ~0.93 g/g. Among the strains screened for their abilities to produce acetate and lactate from lactose, Clostridium thermolacticum DSM 2910 was found to produce large amounts of lactate and acetate. However, it also produced significant amounts of ethanol, CO and H . 2 2The lactate yield was affected by cell growth. During the exponential phase, acetate, ethanol, CO and H were the main products of fermentation with an equimolar acetate/ethanol ratio, 2 2whereas 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. thermoautotrophica, lactose was first converted mainly to lactic acid, then to acetic acid, with a zero residual lactic acid ...

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Publié par	Fyon
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Screening of anaerobic thermophilic bacteria 43 ___________________________________________________________________________ 3 SCREENING OF ANAEROBIC THERMOPHILIC BACTERIA 3.1 Summary 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 Moorella thermoacetica DSM 2955 were able to convert lactate to acetate at thermophilic temperatures with a yield of ς0.93 g/g. Among the strains screened for their abilities to produce acetate and lactate from lactose, Clostridium thermolacticum DSM 2910 was found to produce large amounts of lactate and acetate. However, it also produced significant amounts of ethanol, CO2 and H2. The lactate yield was affected by cell growth. During the exponential phase, acetate, ethanol, CO2 and H2 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. thermoautotrophica, 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.

Screening of anaerobic thermophilic bacteria 44 ___________________________________________________________________________ 3.2 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 processing. 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 (Käppeli et al. 1981). Because of its lack of protein, it is unsuitable for animal or human feeding. It has a high chemical oxygen demand of 57 to 65 g/L or even higher, depending on the cheese manufacturing process and is a major disposal problem of overloading to sewage treatment plants. This lactose source, being directly fermentable 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 production of acetic acid. Anaerobic acetogenesis conserves all the carbon of glucose in the product acetic acid, thus increasing overall yield per glucose molecule by 50% over the aerobic vinegar process (Busche 1991). Acetic acid production from glucose by Moorella thermoacetica under thermophilic conditions appears to be feasible (Shah and Cheryan 1995). With 45 g/L of glucose in the feed of a fed-batch bioreactor and a two-stage CSTR, the productivity and the concentration of acetic acid are 1.12 g/L×h and 38 g/L, respectively. Although most thermophilic acetogens can convert glucose to acetate with a product yield as high as ς90% (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 presence 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 permeate. The former is a homolactic bacterium, which converts lactose to lactate, and the latter can produce acetate from lactate. A new fermentation 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 and an overall productivity of 1.23 g/L×h were obtained. However, a thermophilic fermentation process could be more interesting, since it has generally a higher production rate, should be more resistant to contamination and more convenient to maintain anaerobic conditions required for acetogens. In this work, several potential ways for lactose fermentation to acetic acid under anaerobic thermophilic conditions (~60°C) were studied. Different heterofermentative and acetogenic bacteria were evaluated for their potential use to produce acetate, and a co-culture of two bacteria, Clostridium thermolacticum and Moorella thermoautotrophica, was found to give high acetate yield from lactose.

Screening of anaerobic thermophilic bacteria 45 ___________________________________________________________________________ 3.3 Materials and methods 3.3.1 Microorganisms The acetogenic and heterofermentative bacteria used in this study are listed in Tables 3.2 and 3.3, respectively. The freeze-dried strains were first hydrated in a minimal volume of fresh culture medium in an anaerobic chamber, and then transferred 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. thermolacticum 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 Rijssel et al. 1992, respectively. 3.3.2 Culture media Each bacterial strain was cultivated in the medium as specified in the DSM or ATCC catalogues. 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): K2HPO4, 0.348 g; KH2PO4, 0.227 g; NH4Cl, 2.5 g; NaCl, 2.25 g; FeSO4∙7H2O, 0.002 g; yeast extract (Difco), 2 g; resazurin, 0.001 g; trace element solution, 1 mL. The trace element solution SL-10 (see medium 320 described in the DSM catalogue) contained (per liter in 0.077 M HCl): FeCl2∙4H2O, 1.5 g; ZnCl2, 70 mg; MnCl2∙4H2O, 100 mg; H3BO4, 6 mg; CoCl2∙6H2O, 190 mg; CuCl2∙2H2O, 2 mg; NiCl2∙6H2O, 24 mg; Na2MoO4∙2H2O, 36 mg. Each serum bottle (1-liter) containing 250 mL of the basal medium was flushed with 20% CO2/ 80% N2 gas to remove oxygen, then autoclaved at 121°C for 35 minutes. After autoclaving, additional nutrients contained in a concentrated solution were added to the basal medium, by passing through a microfilter (0.45-mm pore size), to the following final concentrations (per liter of basal medium): 0.5 g MgSO4∙7H2O, 0.25 g CaCl2∙2H2O, 4.5 g KHCO3, 0.3 g cysteine-HCl∙H2O, 0.3 g Na2S∙9H2O, 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 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 CO2 atmosphere. The vitamin solution (see medium 141 in the DSM catalogue) contained (in mg/L): 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 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 sterilization conditions without the risk of cross contamination. However, for the stock cultures, media containing all components were autoclaved for 45 min at 121°C to

Screening of anaerobic thermophilic bacteria 46 ___________________________________________________________________________ ensure complete sterilization. Any medium components that were heat labile were sterilized with a sterile 0.2 mm filter. 3.3.3 Batch culture fermentations All batch fermentation studies were performed in 1-liter screw-capped serum bottles, with 250 ml of medium, and fitted with gas-impermeable black butyl rubber septa under anaerobic and non-controlled pH conditions, in a constant temperature incubator (58∼C, agitation speed: 100 rpm). Fifteen milliliters of a spore or cell (in the exponential 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 syringes throughout the batch fermentation for optical density (OD) reading, pH measurement, and HPLC analysis. 3.3.4 Analytical techniques Lactose, 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 (see Appendix 3.1). 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 the 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 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 (H2 or CO2) 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: 3Total pressure [Pa]×Volumegasm1 Percentage ××H2 or CO2 8.31J×mol%1×K%1×Temperature [K]Volumemedium[L] Cell density was monitored by measuring the optical density at 650 nm (OD650) in a spectrophotometer (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 (see Appendix 3.2).

Screening of anaerobic thermophilic bacteria 47 ___________________________________________________________________________ 3.4 Results 3.4.1 Screening The potential pathways for acetic acid production from lactose fermentation is shown on Table 3.1. Two major groups of bacteria, including acetogenic and heterofermentative bacteria, that might be involved in the acetic acid fermentation were screened (Tables 3.2 and 3.3). Table 3.1. Potential pathways for acetic acid production from lactose fermentation. Step 1 Step 2 (1) Enzyme hydrolysis Homoacetic fermentation Lactose | glucose + galactose | acetate (2) Homolactic acid fermentation Homoacetic fermentation Lactose | lactate | acetate (3) Heterolactic acid fermentation Homoacetic fermentation Lactose | lactate, acetate, ethanol, CO2, H2 | acetate Table 3.2. Screening results of various thermophilic acetogens cultivated in media containing galactose or lactate as sole carbon source. Species Strain Acetate yield Acetate yield from galactose from lactate (mol/mol) (mol/mol) Calorimator fervidus DSM 5463T - - Acetogenium kivui DSM 2030T - - Acetomicrobium flavidum DSM 20664T - - Moorella thermoacetica DSM 2955T - 1.40-1.46 DSM 521T - - DSM 6867T - - DSM 39073T - - ATCC 34490T - - ATCC 31490T - - ATCC 39289T - - Moorella thermoautotrophica DSM 7417T - 1.38-1.46 DSM 1974T 2.0-2.5 - - : no growth

Table 3.3. Summary of screening results for various thermophilic heterofermentative bacteria grown on milk permeate (see Appendix 3.3). Species Clostridium Thermoanaerobacter Thermoanaerobacter Thermoanaerobacter Thermoanaerobacter Thermoanaerobacterium thermTo lacticum T brockii ssp broTc kii EthT anolicus T finnii T thermohTy drosulfuricus T thermosaccharoTl yticum Strain DSM 2910DSM 2911DSM 1457DSM 2246DSM 2355DSM 3389DSM 2247DSM 567DSM 571From the references Le Ruyet et al., 1984, 1985 Zeikus et al., 1979 Wiegel and Ljungdahl, 1981 Schmid et al. 1986 Wiegel et al., 1979 Wiegel 1992 Zeikus et al., 1980 Hollaus and Klaushofer, 1973 Temperature range (°C) 50-70 50-70 35-85 37-78 40-75 37-78 35-67 (optimal temperature) (60-65) (65) (65-70) (69) (65) (67-69) (55) PH range for growth 6.0-7.8 6.0-7.8 5.5-9.5 4.4-9.8 5.5-9.2 7.0-8.5 (optimal pH for growth) (7.0-7.2) (7.2-7.4) (7.5) (5.8-8.5) (6.5-6.8) (6.9-7.5) From the present study Lactose fermented (mmol./L) 13.04 15.02 25.57 31.42 29.74 20.02 42.65 35.38 56.50 Temperature (°C) 60 65 65 65 65 65 65 65 60 Initial pH 7.68 7.42 7.40 7.57 7.28 7.41 7.52 7.77 7.70 Final pH 5.90 5.84 4.76 4.71 4.80 4.76 4.70 4.90 4.82 Product yield (mol/mol) Lactate 2.45 2.02 1.14 0.96 1.24 2.38 0.69 0.79 0 Acetate 0.73 1.00 0.19 0.30 0.23 0.26 0.20 0.58 0.29 Ethanol 0.73 1.00 1.76 1.63 2.06 0.98 2.87 1.26 1.38 CO2 1.46 (1) 2.00 (1) 3.94 4.56 4.7 2.33 3.5 4.80 8.41 H2 1.46 (1) 2.00 (1) 0.53 0.27 0.24 0.59 0.34 3.67 7.44 Other fermentation products - - - - - - - - + detected by HPLC but not identified Biomass (by dry we(2i)g ht in g/l) 0.40 0.43 0.98 0.88 1.31 % carbon recovery 97.00 97.00 93.83 94.23 98.00 Ratio mol lactate / mol ethanol 3.36 2.02 0.65 0.59 0.60 (1) : calculated by carbon balance (2) : the percentage of carbon recovery was calculated as the ratio: Total carbon present in all fermentation products / Total carbon in fermented carbon sources. The strains Thermoanaerobacterium saccharolyticum DSM 7060, Thermobacteroides acetoethylicus DSM 2359, Thermanaerobacterium xylanoliticus DSM 7097, Thermotoga maritima DSM 3109, Thermotoga elfii DSM 4359 and Thermotoga neapolitana DSM 9442 have shown no or poor growth on lactose. 09.76.15 917..8578 900..9472 2.43 0.24 0.63 .0 4697.98 0

Screening of various thermophilic bacteria 49 ___________________________________________________________________________ All anaerobic acetogens can utilize glucose and CO2 and H2 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, twelve known thermophilic acetogens were screened for their abilities to ferment galactose. Table 3.2 shows that only Moorella thermoautotrophica DSM 1974 was able to use galactose when this substrate was present as the only source of carbon and energy, producing ς2.5 mol acetic acid per mol of galactose consumed. However, this bacterium fermented only glucose when both glucose and galactose were present in the medium. Consequently, this bacterium was not suitable to produce acetic acid from hydrolyzed milk permeate. Among the twelve acetogens screened, two strains produce acetate from lactate. M. thermoautotrophica DSM 7417 and M. thermoacetica DSM 2955 produced ς1.4 mol acetic acid per mol lactic acid consumed (0.93 g/g). The growth and degradation rates were very similar for both strains: the pH range for cell growth of M. thermoautotrophica was between 5.0 and 7.8, with an optimal pH at ς6.5 whereas it was at ς7.0 for M. thermoacetica. 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 convert lactose to lactate like Lactobacillus bulgaricus, Bifidobacterium thermophilum, L. lactis and L. helveticus; there is only one known thermophilic lactic acid-producing bacterium, Streptococcus (Lactobacillus) thermophilus. However, this bacterium, though it can grow at 50°C, is usually used in optimal conditions at mesophilic temperature (45°C) in co-culture with the mesophilic lactic bacterium Lactobacillus helveticus (Boyaval et al. 1988). Therefore, various anaerobic thermophilic strains, belonging to the saccharolytic or cellulolytic group of bacteria, were thus screened for their abilities to produce acetic acid from lactose present in milk permeate. Results are summarized in Table 3.3: all were obtained from batch fermentation experiments without pH control or any other attempt to optimize the fermentation conditions. Among these strains, there was no thermophilic homolactic bacterium, and the main fermentation products were lactate, acetate, ethanol, CO2 and H2. As can be seen in Table 3.3, Clostridium thermolacticum DSM 2910 appears to be an appropriate strain for lactate production on account of its high lactate yield (2.45 mol/mol lactose fermented) and high lactate/ethanol ratio (3.36 mol/mol). This bacterium had an optimal growth temperature at ς60 ∼C and growth pH range between 6.0 and 7.8. 3.4.2 Fermentation of C. thermolacticum on lactose To further evaluate the fermentation way to produce acetate from lactose via lactate, H2 and CO2 using heterofermentative and acetogenic bacteria, detailed fermentation kinetics were studied and the results are reported here. Figure 3.1 shows typical kinetics of fermentation of C. thermolacticum grown on lactose. Products from this fermentation included lactate, acetate, ethanol, CO2 and H2. In such batch cultures, cell growth stopped when only ~18 mmol/L 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 ς5.9 when cell growth stopped. During the exponential phase of growth, acetate, ethanol, CO2 and H2 were produced, while lactate formation was relatively small and was delayed. However, neither ethanol nor acetate was produced once cells reached the stationary phase,

Screening of various thermophilic bacteria 50 ___________________________________________________________________________ indicating that their production was growth-associated. On the other hand, more lactate was produced in the stationary phase. The drop of pH generally coincided with acids production. It was also obvious that lactose was hydrolyzed to glucose and galactose, 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 balance calculation, about 97% of the lactose fermented 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), acetate (1), ethanol (1), CO2 (5), H2 (5). The pattern of growth and fermentation in a medium containing 25 mmol/L lactose from milk permeate is shown in Figure 3.2. Products from this fermentation included lactate, acetate, ethanol, and CO2 and H2 (not shown). In this batch culture, the fermentation almost stopped when 13 mmol/L 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 (CO2 and H2) were produced in this batch as compared to the previous one (Figure 3.1). The final product molar ratio in this batch was approximately: lactate (3), acetate (1), ethanol (1), CO2 (2), H2 (2). Because of its content in phosphate (~1.5 g/L), 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.

Screening of various thermophilic bacteria 51 ___________________________________________________________________________ 104021100806040CO2H2200020405404533025201510500Lactose876pH546080100120Cell12..10Lactate0.8Ethanol0.6AcetateGalactose0.40.2Glucose0.020406080100120Time (h)Figure 3.1. 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).

Screening of various thermophilic bacteria 52 ___________________________________________________________________________ 8765050Cell5330Lactose5202511050050010Hp105200250.08Lactate06.0.4GalactoseGlucoseEthanolAcetate0.20100Time (h)150200250Figure 3.2. 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).

Screening of various thermophilic bacteria 53 ___________________________________________________________________________ 3.4.3 Acetogenic fermentation of M. thermoautotrophica on lactate M. thermoautotrophica DSM 7417 homofermentatively converted lactate to acetate at thermophilic temperature (50-65°C) and at pH between 5.5-7.7 (Figure 3.3). Approximately 0.93 g of acetic acid was formed from each gram of lactic acid (Figure 3.4). 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. The pH effect on bacterial growth may be directly attributed to the H+ in the medium. The hydrogen ions may inhibit one metabolic reaction that is the limiting step and thus limit +the growth rate of the culture. In general, the effect of H on the specific growth rate may be represented as follows: m01m 1#H#KH#KOHH# where m0, KH, and KOH are the rate constants. In this equation, H+ is considered as a substrate at alkaline pH (low [H+]), but as an inhibitor at acidic pH (high [H+]). When this equation was fitted with the data at pH between 5.5 and 7.7 using a nonlinear regression method, the best values found for the rate constants were m0 = 0.06, KH = 8.10-7 M, and KOH = 5.4.10-8 M. Since KOH is very small as compared with KH, the KOH term in the equation can be neglected when the medium pH is around 7.0 or lower. This reduces the equation to: m1m0KH##HKH