Anaerobic Digestion: Microbial and Biochemical Aspects of Volatile Acid Production. J. L. Uribelarrea and A. Pareilleux. D6partement de G6nie Biochimique et ...
a;Applied Microbiologyand Biotechnology
European J Appl Microbiol Biotechnol (1981) 12:118-122
9 Spfinger-Veflag.1981
Anaerobic Digestion: Microbial and Biochemical Aspects of Volatile Acid Production J. L. Uribelarrea and A. Pareilleux D6partement de G6nie Biochimique et Alimentaire, ERA-CNRS n~ 879, Institut National des Sciences Appliqu~es, Avenue de Rangueil, F-31077 Toulouse Cedex, France
Summary. The production of organic acids has been tested with bacterial flora selected from a municipal sludge digestor. In order to elucidate the basic mechanisms by which glucose is converted to volatile fatty acids, the examination of non-methanogenic bacteria was attempted. Both lactate-producers and lactate-utilizers were found among these bacteria. When mixed isolates were used as the inoculum, the accumulation of lactic acid and its further conversion to propionic and butyric acids was demonstrated at a carbon conversion rate of about 0.75. It is therefore suggested that this metabolic sequence may occur as a normal process in acidogenic fermentation, which is the first step in anaerobic digestion.
Introduction Usually, anaerobic digestion is thought of as a two-stage process consisting of an acid-forming phase and a methaneforming phase. During the first, organic matter is decomposed into volatile fatty acids, CO2 and hydrogen. These species are then converted to methane, CO2 and other reduced compounds by a methanogenic fermentation (Pohland and Gosh 1971). It has been suggested that anaerobic fermentation should be carried out in two steps, the first being the formation of volatile acids and the second the formation of methane (Andrews 1971 ; Gosh et al. 1975). Most research in this field has been devoted to empirical means of exploiting anaerobic digestion of wastes, rather than to the elucidation of the basic mechanisms by which substrates are converted in products by various microbial species. Hobson and Shaw (1974) made a survey of the anaerobic and facultatively anaerobic bacteria present in piggery wastes. Biselle et al. (1975) reported the existence of 22 nonmethanogenic microorganisms able to convert different carbon sources (cellulose, 0171-1741/81/0012/0118/$01.00
proteins, carbohydrates etc.) into volatile fatty acids. Ueki et al. (1978) have presumptively identified and characterised the distribution of anaerobic bacteria which are active in the decomposition of wastes of domestic animals. The isolation and enumeration of lactate-utilizers has also been reported (Ueki et al. 1980a, b). In the present report, biological and metabolic interactions concerning the acid forming-step of the anaerobic fermentation of glucose are investigated.
Materials and Methods All experiments were carried out in a 3 l fermentor, inoculated with 300 mt of a precuRu~ed medium, under controlled temperature and pH conditions. After sterilization, the medium was deoxygenated with a stream of CO2. The composition of the culture medium was: Na2HPO4 9 12H20, 0.42; NaH2PO4 9 2H20, 0.18; (NH4)2HPO4 " 2H20, 2; K2HPO4 92H20, 0.5; MgSO4 9 2H20, 0.4; CaC12, 0.03; KC1, 0.08; FeSO4, 0.08 and yeast extract, 0.3 g per litre. The cell mass was determined by dry weight measurements after filtration on 0.22/~m pore size millipore filters. The residual glucose was determined by H.P.L.C. using a NH2-silica column with acetonitrile : water (80 : 20) as the elution solvent. The lactic acid concentration was determined by an enzymatic method with lactate dehydrogenase. Volatile organic acids were determined by gas chromatography analysis, according to the method of Bricknell et al. (1975), using a column packed with Porapack Q 80.100 mesh. The samples were acidified with 1 N HC1 before chromatography. The volume of the gas evolved during the reaction was measured volumetrically. Its composition was determined by gas chromatography according to the method of Deans et al. (1971), using Porapack Q and molecular sieve columns. The total bacterial flora used as an inoculum was selected from a municipal sewage digestor as described by De la Torre and Goma (1981). The isolation of microorganisms was attempted using Brewer anaerobic agar. The aerotolerance of each isolate was tested on fluid thioglycolate medium and trypticase soymeal agar plates. Clostridia were detected and isolated on D.R.C. medium according to the method of Freame and Fitzpatrick (1971). The purity was determined on the basis of bacterial morphology
J. L. Uribelarrea and A. Pareilleux: Anearobic Digestion
119
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Fig. l Time course of a batch fermentation (inocutum; total bacterial population: substrate; glucose, 45 g/l: temperature; 40 ~ and pH 6). The dry weight (g/l); - ~ - X: glucose (g/l); - ~ - S: products (g/l);-~ZPi and gas production (1/i); - A - are shown
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(microscopic observation) and colony form. The presumptive identification of each isolate was confirmed by determining their fermentation products which are characteristic of microbial gro ups. Lactate-utilizers were selected by enrichment batch culture using the above mentionned mineral medium and L-lactic acid, as the sole carbon source. Some microorganisms of this group were judged to belong to sulphate-reducing bacteria, according to the method of Plankhurst (1971).
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Fig. 2. Relationship between dP/dt and dS/dt during the second steop of the fermentation: - e - P2, acetic acid; - o - P4, butyric acid and - 0 - Zpi, total volatile acids
Table 1. Yields (R) and carbon conversion rates (Rc) for the production of volatile acids. Acetic acid is denoted by P2 and butyric acid by P4 Yields (g/g) or carbon conversion rates
RP2 RcP2 RP4 RcP4 R (P2 + P4) Rc (P2 + P4) Rgas
Temperature 40 ~
30 ~
0.09 0.09 0.39 0.54 0.48 0.63 0.34
0. 3 0.3 0.22 0.38 0.52 0.60 0.032
Batch cultures of the total bacterial population were grown at either 30 ~ or 40 ~ pH 6. The initial substrate concentration was 45 g/1 glucose. As an example, the time course of a batch cultivation at 40 ~ is shown in Fig. 1. The cell mass increased until approximately 50% of the glucose had been consumed and then remained unchanged. Gas production was associated with the formation of acetic and butyric acids. It is notworthy that even after the increase in biomass ceased, the production of volatile acids continued. Assuming that all of the substrate was reduced during the second fermentation step, the yields for gas formation and for organic acid production can be determined from a linear relationship between dP/dt and dS/dt as shown in Fig. 2. The values calculated for the two operational temperatures are summarized in Table 1. The yields of butyric acid and the amounts of gas produced were higher at 40 ~ than at 30 ~ but the carbon conversion rates for the total production of volatile acids were in the same range. The following equation for organic acid production;
aC6Ht206 -+ bC2H402 + cC4H802 + dCO2 + e H 2 + f H 2 0 ,
120
J. L. Uribelarrea and A. Pareilleux: Anaerobic Digestion
Table 2. Experimental and calculated stoichiometric coefficients Determination method
Experimental Gas production Substrate consumption E. M. metabolic pathway
Coefficients a (Substrate)
d (CO2)
e (H2)
d+e (Total gas)
1 1 0.87 0.93
2.86 1.44 1.87
2.92 1.28 1.6
2.73 5.18 2.73 3.47
tion of the acidogenic flora occurred due to the environmental conditions. Several bacterial groups were then isolated and characterized from the total flora. Both obligate and facultative 1.5 anaerobic bacteria were isolated. As an initial identification, the isolated bacterial groups were characterized according to their glucose fermentation end products: bu,1 1 ~ tylene glycol; lactic acid (homo- and heterofermentation); butyric acid-butanol-acetone (Clostridia) or acetic, lactic and butyric acids. .0.5 0.6 A lactate-producing strain (homofermenter) converted 26.5 g/1 glucose to 25.4 g/1 lactic acid (L-lactic acid 65%, D-lactic acid 35%) which is a carbon conversion rate of 0.96. When an isolated Clostridium was used, production 4 0 t 6 0 Ch? 8 0 20 0 of ethanol, acetic and butyric acid was observed and the maximum growth rate of this strain was 1.3 h - 1 (subFig. 3. Volatile acid production by lactate-utilizers (substrate; L-lactic acid, 16.8 g/l: temperature; 35 ~ pH 6): -zx- X, dry strate, glucose; 40 ~ pH 6). weight (g/l); - = - S, L-lactic acid (g/l); - e - P2, acetic acid (g/l); The organic yields were in the same range as obtained - ~ - P3, propionic acid (g]l) - o - P4, butyric acid (g/l) and with the total bacterial flora and corresponded to a car-A-, gas production (1/1) bon conversion rate of 0.66. Nevertheless, the gas production (0.5 1/g glucose) and its average composition (CO2, 45%; H2, 55%) were in good agreement with the classical, metabolic pathways of Clostridia. Since lactic acid proallows comparison of the different coefficients derived duction could occur without any loss of carbon as CO2, from experimental results or calculated in three possible it appears that lactic acid is the supposed accumulated ways: Firstly, the gas production corresponding to the compound. In such a case, the carbon conversion rate amount of substrate consumed; secondly, the theoretical from glucose to acids may be higher than 0.66 (see Table substrate consumption, deduced from the observed gas 2) in the presence of the total microbial population. We production and thirdly, the gas production and the subthen considered the possibility that a partial conversion strate consumption when this stoichiometry describes of lactic acid to other compounds occurred. Lactate-utithe Embden-Meyerhoff metabolic pathway followed by lizers selected by enrichment cultivation converted L-lacpyruvate decarboxylation. tic acid (16.8 g/l) to acetic acid (0.3 g/l), propionic acid These values, for a cultivation temperature of 40 ~ (4.6 g/l) and butyric acid (6.1 g/l) (Fig. 3). The gas proare listed in Table 2. The amount of sugar consumed is duction (CO2; H2 S traces) was 0.105 1/g L-lactic acid. higher than the amount which is theoretically necessary The carbon conversion rate into volatile organic acids to produce the observed levels of gas and acids (Table 2). was 0.85 and it was clearly demonstrated that propionic We therefore assume that product accumulation occurs acid production was associated with lactic acid conversion. and moreover that a metabolic pathway other than that Based on H2 S production, ethanol utilization and other previously considered is involved in the acidogenic procharacteristics, some of these lactate-utilizers are thought cess. Furthermore, during successive batch cultivation to belong to the sulphate-reducer group. (40 ~ pH 6) a significant change in the profile of organIn order to relate the types of microbes present to bioic acids was observed and the carbon conversion rate of glucose increased to 0.76. It may be concluded that selec- chemical aspects of the conversion, a glucose fermenta-
J. L. Uribelarrea and A. Pareilleux: Anaerobic Digestion
121
40-
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Fig. 4 A, B
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Glucose conversion by a reconstituted population (temperature, 35 ~ pH 6). A - • - X, dry weight (g/I); - D - S, glucose (g/l);--0- 2P, total products (g/l) and - A - gas production (1/1). B - = - , D-L-lactic acid (g/l); -| ethanol (g/l); - e - P2, acetic acid (g/l); - e - P3, propionic acid (g/l) and - o - P4, butyric acid (g/l)
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where: R x , cell yield (g/g) and Rp, total product yield (g/g).
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As shown in Fig. 5 (linear relationship between - d S / d X and dP/dX), it follows that the biomass yield and the product formation can be expressed as the intercept and the slope, i.e.: R x , 0.75 g/g glucose and Rp, 0.61 g/g glucose. This value of Rp corresponded to a carbon conversion rate; Rcp , 0.75 which is in good agreement with the previous one.
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acid (0.74 g/l) and butyric acid (13.95 g/l) after 20 h. In the same time the biomass concentration reached 6.5 g/1. Then, as expected, lactic acid was converted to propionic and butyric acids and the ethanol concentration decreased. It was noticed that the rate of propionate production was constant during the glucose utilization phase while the concentration of acetic and butyric acids increased exponentially. A growth inhibition of lactate-utilizers by glucose may therefore occur when lactate is used as carbon source. The sugar conversion can be described by the following equation;
dP/dX
Fig. 5. Relationship between -dS/dX and dP/dX; intercept R X = 0.75 g/g glucose and slope Rp = 0,61 g/g
tion was performed with a mixture of the singly cultivated isolates as the inoculum: lactate-producers, lactate utilizers and butyric Clostridia. As shown in Fig. 4a, b all of glucose (45 g/l) was converted to ethanol (0.4 g/l), acetic acid (5.34 g/l), D and L lactic acid (0.87 g/l), propionic
Discussion It is accepted that it is quite difficult to understand microbial ecosystems, especially in sewage digestor fluids. One approach is to study the end products of the degradation of various substrates (Chynoweth and Mah 1971 ; Gosh et al. 1975). With this approach many authors think that it is significant to analyse bacterial flora by the cultural method
122 (Hobson and Shaw 1974; Ueki et al. 1978). However, analysis of the bacteria present in a sewage digestor requires their isolation with special techniques. Studies of the isolates by the cultural method m a y be invalid because strains are often interdependant in mixed cultures. Moreover, the bacteria present in anaerobic digestors depend to some extent on the type o f input waste and the numbers and types of bacteria found reflect the different available substrates. The foregoing was allowed for in experiments to investigate microbial, metabolic and kinetic aspects of glucose fermentation by the microbial flora of a digestor. Using total flora, stoichiometric and carbon balance analysis suggested that product accumulation occurred during the conversion of glucose to volatile organic acids and furthermore that an indirect metabolic p a t h w a y might be involved in the process. Initial characterization of the bacterial flora was a t t e m p t e d using classical cultural methods which showed that most o f the microbial types described previously were present in the digestor fluid. No strains were found able to convert hexoses to volatile fatty acids by a metabolic pathway which did not involve pyrurate decarboxylation. Facultatively anaerobic bacteria were surprisingly the most numerous and some o f these were lactate producers. The demonstration that lactateutilizers convert lactic acid to volatile organic acids agrees well with the fact that this acid is often not detected in digestor fluids (Ueki and al. 1978). As expected, using a reconstituted mixed population, the accumulation of lactic acid from glucose and its subsequent conversion to propionic and butyric acids was demonstrated. Such a metabolic sequence which depends on the glucose concentration because the growth of lactate-utilizers is suppressed by glucose, leads to the high carbon conversion rates observed by De la Torre and Goma (1981). These results suggest that the formation and subsequent conversion of lactic acid m a y occur as a normal process in digestors. It is believed that lactate-producing and lactate-utilizing bacteria play an important role in the fermentation process during sewage digestion (Ueki et al. 1980a, b). However, since many lactate-utilizers require CO2 for their growth (Hungate 1966; Dehority 1971) the metabolic pathway by which lactic acid is converted remains unknown.
J.L. Uribelarrea and A. Pareilleux: Anaerobic Digestion References Andrews JF (1971) Kinetics model of biological waste treatment processes. Bioteehnol Bioeng Syrup 2:5-33 Biselle C, Koertreich M, Scholl M, Spewak P (1975) Urban trash methanation background for a proof of concentration experiment. MITRE Report N~ MTR 6856 Bricknell KS, Sutter VL, Finegold SM (1975) Detection and identification of anaerobic bacteria. In: Mitruka BM (ed) Gas chromatographic applications in microbiology and medicine. J. Wiley & Sons, New York, London, pp 251-277 Chynoweth DP, Mah RA (1971) Volatil acid formation in sludge digestion. In: Pohland FG (ed) Anaerobic biological treatment process. Ann Chem Soc, pp 41-54 Deans DR, Huckle MT, Peterson RM (1971) A new column system for isothermal gaz chromatographic analysis of light gazes employing a column switch technique. Chromatographia 4: 279285 Dehority BA (1971) Carbon dioxide requirement of various species ofrumen bacteria. J Bacteriol 105:70-76 De La Torre I, Goma G (1981) Characterization of anaerobic microbial culture with high acidogenie activity. Biotechnol Bioeng 23:185-199 Freame B, Fitzpatrick BWF (1971) The use of differential Clostridium medium and enumeration of Clostridia from food. In: Shapton DA, Board RG (eds) Isolation of anaerobes. Academic Press, New York, pp 49-54 Gosh S, Conrad SR, Klass DL (1975) Anaerobic acidogenesis of waste water sludge. J Water Poll Control 47:30-45 Hobson PN, Shaw BG (1974) The bacterial population of piggery waste anaerobic digestors. Water Res 8:507-516 Hungate RE (1966) In: The tureen and its microbes. Academic Press, New York, pp 20-21 Plankhurst ES (1971) The isolation and enumeration of sulphate reducing bacteria. In: Shapton DA, Board RG (eds) Isolation of anaerobes. Academic Press, New York, pp 223-238 Pohland FG, Gosh S (1971) Developments in anaerobic treatment processes. Biotechnol Bioeng Syrup 2: 85-106 Ueki A, Miyagawa E, Minato H, Azuma T, Suto T (1978)Enumeration and isolation of anaerobic bacteria in sewage digestor fluids. J Gen Appl Microbiol 24:317-332 Ueki A, Minato H, Azuma R, Suto T (1980a) Enumeration and isolation of anaerobic bacteria in sewage digestor fluids: isoiation of lactate-utilizers. J Gen Appl Microbiol 26:15-24 Ueki A, Minato H, Azuma R, Suto T (1980b) Enumeration and isolation of anaerobic bacteria in sewage digestor fluids: enumeration of sulfato-reducers by the anaerobic roll tube method. J Gen Appl Microbiol 26:25-35
Received December 12, 1980
