lactate and ethanol as intermediates in two-phase anaerobic digestion

Lactate and Ethanol as Intermediates in Two-Phase Anaerobic Digestion

P. PIPYN and W. VERSTRAETE, Laboratory of Microbial Ecology, State University of Ghent, Coupure 533, 9000 Gent, Belgium

Summary The thermodynamics of the various anaerobic digestion patterns of hexose to methane

are compared. It appears that by directing the hexose-hydrolysis phase towards ethanol and lactic acid production, methanogenesis can be enhanced because the syntrophic bacteria are allocated more potentially available energy. This hypothesis was confirmed in a series of laboratory test runs. They revealed that lactic acid and ethanol as intermediates, in comparison to lower volatile fatty acids, give rise to a considerably higher effluent quality and a slightly larger biogas production.

INTRODUCTION

The first step in the conversion of polymers such as carbohydrates, fats, and proteins to methane, is hydrolysis. This is achieved by various fermentative bacteria, each of them having a very specific catabolic ac- tivity. The second step is brought about by the obligate hydrogen-producing acetogenic bacteria. I They use volatile fatty acids (VFA), ethanol, and lactic acid produced by the first group of bacteria and convert it into acetic acid, carbon dioxide, and hydrogen. Finally, the latter products are used by the methanogenic bacteria. The question thus arises whether the methanogenic bacteria and the methane formation can be favored by intervening in the relationships between the different microbial groups.

In this context, a number of authors have proposed to engineer anaerobic digestion in two phases, one phase under conditions optimal for acid formation, and one under conditions optimal for methane prod~ction.*-~ In this work, the biochemical and energetic interdependence of the different anaerobic trophic groups are analyzed in relation to the most optimal type of hexose-hydrolysis fermentation.

The anaerobic degradation of carbohydrates is relatively well under- stood in terms of biochemistry and thermodynamics. The EMP and HMP are the most important pathways for hexose degradation in anaerobic bacteria and pyruvate takes the control position, from where alcoholic, lactic acid, or volatile fatty acid fermentations start.6 Comparison of the energetics of the biochemical reactions leading to the formation of meth-

Biotechnology and Bioengineering, Vol. XXIII, Pp. 1145-1 154 (1981) 0 1981 John Wiley & Sons, Inc. CCC 0006-3592/81/053 145-10$01.00

1146 PIPYN AND VERSTRAETE

ane from glucose through different metabolic pathways permits one to determine the most energetically favorable reaction for the so-called syntrophic microorganisms, that is, the obligate hydrogen-producing acetogenic bacteria living in close association with the methanogenic bacteria. The different fermentation patterns are thermodynamically evaluated by means of the values of the energy content of the fermentation products reported by Thauer et The energy values relate to standard physiological conditions (AGO'), that is, the pH 7 and the concentrations of substrates and products are 1 mol/kg. H2 is in the gaseous state, all other substances are in aqueous solution.

Thermodynamic Considerations

From 1 rnol glucose, 2 rnol of acetic acid and 4 rnol of hydrogen can be produced by the fermentative microorganisms [eq. (l)]. Subsequently, the syntrophic methanogenic bacteria produce methane from acetic acid and hydrogen [eqs. (2) and (3)]. The sum reaction is given by eq. (4).

C6H12016 + 4H20+ 2CH3COO- 4- 2HC03- + 4H+ + 4H2 -206.3 kJ (1)

-62.0 kJ (2)

-135.6 kJ (3)

C ~ H I ~ O ~ + 3H20+3CH4 + 3HCO3- + 3H+ -403.9 kJ (4)

2CH3CO0 - + 2H20 + 2CH4 + 2HC03 -

4H2 + HCO3- + H + + CH4 + 3H2O

The fermentation gases of the acid phase [eq. (l)] escape the fermentation medium if no gas recirculation is applied towards the methane reactor. In that case, reaction (3) cannot take place, resulting in a sub- stantial loss of energy for the syntrophic methanogenic bacteria.

From 1 mol glucose, 2 mol propionic acid can also be formed by the fermentative microorganisms [eq. (31. The syntrophic bacteria have to convert propionic acid into acetic acid and hydrogen [eq. (6)] to permit methane formation [eqs. (7) and (8)]. The sum reaction (9) however, is the same as in the previous case.

C6H1206 + 2H2+ 2CH3CH2COO- + 2H20 + 2H+ -358.1 kJ (5)

2CH3CH2COO- + 6H20+ 2CH3COO- + 2HC03- + 2H+ + 6H2 + 152.2 kJ (6)

2CH3COO- + 2H20 + 2CH4 + 2HC03- -62.0 kJ (7)

4H2 + HC03- + H' +CH4 + 3H20 -135.6 kJ (8)

-403.5 kJ (9) C6H1206 + 3H20 +3CH4 + 3HC03- + 3H'

The fermentation of 1 rnol glucose by fermentative microorganisms can also lead to the production of 1 rnol butyric acid [reaction (lo)] which

LACTATE AND ETHANOL IN ANAEROBIC DIGESTION I147

must be converted into acetic acid and hydrogen [eq. (1 l)] and further into methane [eqs. (12) and (13)] by the syntrophic methanogenic bacteria. Reaction (14) represents the sum.

C ~ H ~ Z O ~ + 2H20+ CH3CH2CH2COO- + 2HC03- + 3H' + 2H2 -254.6 kJ (10)

+48.1 kJ (1 1) CH3CH2CH2COO- + 2HZO- 2CH3COO- + H + + 2H2

2CH3COO- + 2H20+2CH4 + 2HC03-

4Hz + HC03- + H' + CH4 + 3H20

- 62.0 kJ (12)

- 135.6 kJ (13)

-404.1 kJ (14) C6H1206 + 3H20+3CH4 + 3HC03- + 3H+

An alcoholic fermentation of glucose in the first phase is possible as well [eq. (IS)]. A subsequent conversion of ethanol into acetic acid and hydrogen [eq. (16)] is needed before any methane production can start [eq. (17)l. The sum (19) is still the same as in the three previous cases.

C6H1206 + 2Hz0+2C2HSOH + 2HC03- + 2H' -225.9 kJ (15)

2CzHSOH + 2HZO+2CH,COO- + 2H+ + 4H2 + 19.2 kJ (16)

2CH3COO- + 2H20- 2CH4 + 2HCO3-

4H2 + HC03- + H++CH4 + 3H20

-62.0 kJ (17)

- 135.6 kJ (18)

-404.3 kJ (19) C6H12O6 + 3H2O + 3CH4 + 3H20

A lactic acid fermentation by the fermentative microorganisms results in the production of 2 mol lactic acid per mol glucose [eq. (20)l. After lactic acid is converted to acetic acid and hydrogen in the second phase [eq. (21)], methane production can start [eqs. (22) and (23)l. The sum reaction is given by reaction (24).

C6H1206+ 2CH3CHOHCOO- + 2H' - 198.3 kJ (20)

2CH3CHOHCOO- + 2CH3COO- + 2HC03- + 2H+ + 4Hz -8.4 kJ (21)

-62.0 kJ (22) - 135.6 kJ (23)

-404.3 kJ (24)

2CH3COO- + 2H20+ 2CH4 + 2HC03- 4H2 + HC03- + H + + C H 4 + 3H20

C6H1206 f 3H20+ 3CH4 + 3HC03- + 3H'

As can be seen from the free energy change of the different biochemical reactions, the energy quantum remaining for the syntrophic methanogenic bacteria depends strongly on the endproduct of the acid phase (hydrolysis of glucose). Table I summarizes the energy quantum available for the fermentative acidogenic microorganisms on the one hand and for the syntrophic methanogenic bacteria on the other hand. One can see that


TABLE I Distribution of the Total Free Energy Change of the Two-Phase Anaerobic Fermentation

of Glucose to Methane over the Different Microbial Groups

Free energy change available for the syntrophic bacteria as Free energy change

available for the End product acid fermentatives H2 gas" otherwise

phase (%I (%I (96) Acetic acid 51.1 33.6 15.4 Propionic acid 88.1 0.0 11.3 Butyric acid 63.0 16.8 20.2 Ethanol 55.9 0.0 44. I Lactic acid 49.0 0.0 51.1

a Potentially subject to loss if headspace gases are not reintroduced into the methane reactor.

fermentation of glucose to volatile acids is not advantageous for the syntrophic methanogenic bacteria. The lactic acid fermentation, on the other hand, is most profitable for these bacteria. These calculations indicate that it might be possible to improve methane digestion by directing the hexose-hydrolysis phase towards ethanol and lactate production. Indeed, by doing so, the most delicate and rate-determining bacterial groups would be allocated a maximum of potentially available energy (Table 11).

EXPERIMENTAL

In order to verify the theoretical concepts, two fermentation experi- ments were set up. The medium contains per liter of water 5 ml molasses ( ? 70% dry matter) as well as NH4CI and K2HP04 to assure a C : N : P ratio of 100: 6: 1. The PH of the solution is 7.0.

Earlier investigation^^*^ revealed that the application of a physiological stress factor such as, e.g., substrate overload or pH shock from 7 to 4

TABLE I1 Free Energy Change of Methane Production from

Different Fermentation Products

Free energy change AGO' per Substrate mol CH4 produced (kJ)

Acetic acid -31.0 Propionic acid - 32.3 Butyric acid - 32.1

Lactic acid -68.8 Ethanol - 59.5

LACTATE AND ETHANOL IN ANAEROBIC DIGESTION I149

enhanced lactic acid bacteria. Sludge retention or sludge recirculation however leads to the build-up of a microbiota which produces volatile fatty acids.

An ethanol-lactic acid and volatile acid fermentation were set up, each of them under nonaxenic conditions.

Fermentation A: batch lactate fermentation in a 25-liter PVC container; no agitation; no pH correction; hydraulic retention time equal to 24 hr; daily cleaning of the fermentor in order to prevent any important sludge retention.

Fermentation B: batch volatile acid fermentation in a 25-liter PVC container; no agitation; no pH correction; hydraulic retention time equal to 24 hr; partial sludge recirculation.

The second step of the two-phase anaerobic fermentation process, the methane fermentation, is performed in an anaerobic expanded bed reactor. The experimental setup used is a Plexiglas cylinder with a volume of 7 liter. An inverted funnel collects the fermentation gases which are meas- ured by a water displacement device. The influent of the expanded bed reactors is prepared as follows: 7 liter effluent of the acidification phase + 3 liter tap water + 10 ml of a 10% NH4Cl solution + 10 ml of a 3% K2HP04 solution. The pH is adjusted to 7.0 by addition of a 10% NaOH solution. This liquor is fed to the lower part of the upflow units by means of a membrane pump (type Liquid Metronics A1 13-95), and recirculated twice a day over the upflow units. The biogas fermentation is terminated after 24 hr, when gas production no longer occurs. Each test run com- prised several weeks until a stable gas production was reached. The upflow units were inoculated with sludge from a large scale upflow diges- tor. The expanded bed contained between 2.5 and 7.5 cm of sludge blanket.

Lactic acid was determined by means of the Conway microdiffusion method." Samples to be analyzed for volatile fatty acids were acidified to pH 2, extracted with ether and subsequently analyzed using a gas chromatograph (Hewlett-Packard 5700A; column: 15% Carbowax 20 TPA on Chromosorb WAN 80-100 mesh, 1.8 m length, flame ionization detector). I '

Fermentation gases as CH4, C02 , and N2 were determined by gas chromatography with hydrogen as carrier gas (Fisher Gas Partitioner, column: 21 ft (0.25 in.) silica gel + 6.5 ft (0.3 in.) molecular sieve; thermal conductivity detector). The fermentation gases H2 and CH4 were determined by gas chromatography with argon as carrier gas (F&M dual column gas chromatograph; column: 3 in. mo!ecular sieve + 4 ft (0.25 in.) silica gel; thermal conductivity detector).

Ethanol was determined enzymatically.'* The reducing sugars were determined colorimetrically by the method of Hofmann et al. l 3 Sucrose was split up in a-glucose and f3-fructose by the 6-fructosidase enzyme and subsequently quantified as reducing sugar. The chemical oxygen de- mand (COD) was determined by the dichromate m e t h ~ d . ' ~


RESULTS

Table I11 shows the results of the acid fermentations A and B, respectively, each of them followed by a methane fermentation. This table gives the average breakdown of the organic matter (as COD) in the two phases of the fermentation process, the biogas production, and the calculated gas yield coefficient for the methane fermentation.

Fermentation balances are made up for the different acid fermentations (Tables IV and V). These balances indicate that lactic acid was the major metabolite during test run A, in contrast to volatile fatty acids during test run B. Consequently, the production of C 0 2 and H2 was much lower during the former test run, resulting in a lower loss of reducing equivalents from the reactor (Table VII). However, due to the strong pH drop, fermentation A slowed down when about half of the initial amount of sugar was converted (Table IV). Therefore, the procedure for test run A was modified: the reactor was slowly stirred (40 rpm) and the pH was auto- matically corrected to 4.2 when it tended to drop below this value. The results of this test run are given in Tables I11 and VI. In this case, all initial sucrose was fermented. However, the fermentation pattern shifted from homolactic to heterolactic.

TABLE 111 Breakdown of the Organic Matter and Biogas Production during the Test Runs A and A bis (First Phase: Ethanol-Lactate Fermentation) and Test Run B (First Phase: Volatile

Fatty Acid Fermentation)

Fermentation Fermentation A A bis Fermentation B

Mean SD= Nb Mean S D ~ Nb Mean S D ~ Nb

Influent first reactor COD 4520 248 6

Effluent first reactor COD 4022 263 6

COD supernatant (mg/liter)' 3634 354 6 Influent methane reactor COD 2876 262 6

Effluent methane reactor:

(mgiliter)

(mg/liter)

(mg/hter)d

suspended solids 250 43 8

COD supernatant 347 76 8 (mgiliter)

(mg/liter) g COD fermented/day 25.3 - - ml biogas/g COD fermented 303 - - ml biogasig COD removed 344 - - Percent of CHI in biogas 92.5 1.8 3

4460 460 8

3725 385 8

3350 345 8 2656 310 1 1

263 184 I 1

556 160 I 1

21.0 - - 253 - - 320 - - 84.1 4.0 3

4973 364 8

4148 402 8

3405 517 8 3071 417 12

441 268 13

837 107 13

22.3 - - 243 - - 335 - - 87.5 2.4 3

a Standard deviation. Number of observations. After centrifugation. Effluent of the acid phase is diluted with tap water to a choosen conceniration.

LACTATE AND ETHANOL IN ANAEROBIC DIGESTION 1151

TABLE 1V Fermentation Balance for the Acid Formation during Test Run A (Lactic

Acid-Ethanol Fermentation)

mmolil00 mmol Product mmoliliter mmol C3 C

Sucrose fermented 8.8 f 0.5 100 300 Products formed

Lactic acid 12.3 f 2.3 34.9 2 6.5 104.7 2 19.5 Acetic acid 2.1 f 0.5 6.0 t 1.4 12.0 t 2.8 Propionic acid 0.0 f 0.0 0.0 2 0.0 0.0 2 0.0 Butyric acid 0.0 5 0.0 0.0 ? 0.0 0.0 * 0.0 Valeric acid 0.0 2 0.0 0.0 f 0.0 0.0 2 0.0 Ethanol 0.8 f 0.3 2.3 ? 0.9 4.6 ? 1.8

DISCUSSION

Under the nonaxenic conditions of the test runs, two distinct fermentation patterns were observed. At very short cell residence times, a homolactic (test run A) up to heterolactic pattern (test run A bis) was noted. In the latter case, the free energy change remains relatively low:

C6H12O6 + CH3CHOHCOOH + CzHsOH + C02 (25)

AGO’ = -212 kJlr

Accumulation of lactate in anaerobic sludges has been reported to occur when sludges receive shock loadings of g l u c o ~ e . ~ ” ~ The formation of both lactate and ethanol in equimolar amounts is typical for the heterolactic acid bacteria associated for instance with the sauerkraut fermentation. When the reactor was operated with partial sludge recirculation (cell residence times of 3-5 days), a mixture of volatile fatty acids-of which propionate, acetate, and butyrate were the dominant ones-was produced (test run B). When the composition of the fatty acids, as well as the

TABLE V Fermentation Balance for the Acid Formation during Test Run B

(Volatile Acid Fermentation, Partial Sludge Recirculation)

Product mmol/li ter mmol C3 C mmoli 100 mmol

Sucrose fermented 9.7 t 0.7 100 300 Products formed

Lactic acid 0.6 f 0.2 1.5 ? 0.5 4.5 2 1.5 Acetic acid 8.6 f 1.4 22.2 ? 3.6 44.4 f 7.2 Propionic acid 9.4 ? 2.7 24.3 t 7.0 72.9 ? 21.0 Butyric acid 4.0 2 1.3 10.3 ? 3.4 41.2 f 13.6 Valeric acid 2.3 ? 0.4 5.9 -t 1.0 29.5 2 5.0 Ethanol 1.9 t 1.4 4.9 -t 3.6 9.8 2 7.2

I152 PIPY N AND VERSTRAETE

TABLE VI Fermentation Balance for the Acid Formation during Test Run A bis

(Lactate-Ethanol Fermentation with pH Correction)

rnmo1/100 mmol Product mmoliliter mmol C3 C

Sucrose fermented 8.7 ? 1.3 100 300 Products formed

Lactic acid 13.9 t 2.1 40.0 ? 6.0 120.0 t 18.0 Acetic acid 3.2 ? 1.6 9.2 f 4.6 18.4 5 5.2 Propionic acid 0.4 f 0.8 1 . 1 k 2.2 3.3 2 6.6 Butyric acid 0.8 ? 1.2 2.3 t 3.4 9.2 ? 13.6 Valeric acid 0.0 t 0.0 0.0 '' 0.0 0.0 t 0.0 Ethanol 12.5 f 2.6 35.9 k 7.5 71.8 5 15.0

percent COD loss (presumably in the form of H,) is taken into account, the fermentation approximates the following pattern:

+ ICHsCHzCH2COOH + 4HzC03 + 4H2 (26)

AGO' = - 806 kJ/r

or - 269 kJ/mol glucose

The pattern of metabolite formation in the hydrolysis reaction affects the amount of COD lost in the form of hydrogen gas (Table WI). The fact that during test run A bis the losses of H2 almost equalled those in test run B is probably due to the stirring of the mixed liquor. In two- phasic biogas systems, the hydrogen formed in the first reactor can be collected and either used as such or introduced in the methane reactor. From a practical point of view however, it appears advantageous to min- imize its formation and thus maximize the amount of methanogenic substrate introduced in the second reactor.

The pattern of metabolite formation has a more pronounced effect on the build-up of fermentative biomass. The amount of COD converted by

TABLE VII COD Balance for the Acid Fermentation

COD available COD COD converted to the

volatilized to sludge methanogens Fermentation run (%) (%) (%)

Lactate-Ethanol A 11.0 8.6 80.4 A bis 16.4 8.4 75.2

B 16.6 14.9 68.5 Fatty acids

LACTATE AND ETHANOL IN ANAEROBIC DIGESTION 1153

the fermentative bacteria to sludge is 1.7 times larger in the case of B compared to A (Table VII). This relates to the amount of energy available from the ethanol-lactic acid fermentation at one hand and from the fatty acid fermentation on the other hand [eqs. (25) and (26)l.

In view of the fact that the production of fermentative sludge coincides with a reduction of directly available substrate for the methanogens, it is obvious that the first fermentation qualifies as the most preferable one. This is even more so since the fermentative biomass produced tends to go through the methane reactor and thus decreases the quality of the final effluent (Table 111).

The final effluents of test runs A and A bis contain significantly less residual COD than the ones of test run B. It is well known that the removal of volatile acids such as butyrate and propionate depends on the action of the acetogenic hydrogen-producing bacteria. Furthermore, the metab- olism of these acids yields relatively little energy to the syntrophic association (Table 11). It is generally recognized that these products, and particularly propionic acid, tend to persist in the effluent. As a matter of fact, the concentration of propionic acid in the test runs A, A bis, and B amounted to 35, 73, and 95 mdliter, respectively.

A final advantage of the lactate-ethanol fermentation is that it resulted in a minimum of odor nuisance, while the formation of fatty acids ne- cessitated active odor abatement.

Cohen et a].’ have reported for a two-phase digestion with butyrate and acetate as intermediates a 12% loss of substrate in the form of H2 and 11% immobilization in the form of biomass. The latter data are in agree- ment with the theoretical considerations summarized in Tables I and I1 and with the results of these test runs. They indicate that butyrate ranks third to lactate and ethanol as a “feasible” intermediate for two-stage anaerobic digestion. “Feasible” relates in this context to the practical application of wastewater treatment. Indeed, the fermentation pattern considered must be sufficiently competitive to succeed under nonaxenic conditions. Furthermore, it must be reliable and simple to control. To what extent the mixed lactate-ethanol fermentation can successfully be imposed in large scale treatment plants needs further study. It is clear that the extra requirement for pH control implicates additional treatment costs which, for a full-scale treatment plant, will have to be evaluated against the advantages of better effluent quality.

Obviously, two-phase digestion is only warranted in cases where the major part of the initial substrate is not directly accessible for the methanogens. In some instances, the nature of the substrate as well as the constitution of the wastewater permit an active induction of a heterolactic fermentation. The results of this laboratory study indicate that in these instances it might be advantageous to impose this fermentation pattern, particularly with respect to the quality of the final effluent.

The microbial and biochemical mechanisms which give rise to the rapid conversion of ethanol and lactic acid to methane and carbon dioxide


require further elucidation. Most probably, the conversion of lactic acid is on the one hand the result of protocooperative action between homo- acetogenic bacteria converting lactic acid to acetic acid and hydrogen, and on the other hand a methanogenic bacteria growing on the hydrogen and acetate thus produced (Zeikus, personal communication).

CONCLUSION

The two-phase methane fermentation process was optimized by imposing a mixed lactate-ethanol fermentation in the first reactor. Thus 75-80% of the influent COD (present in the form of sugars) could be submitted as directly available substrates to the methanogenic bacteria. When a spontaneous volatile fatty acid fermentation took place, only 68% of the influent COD was available to the methanogens. By imposing a lactate-ethanol over a mixed volatile fatty acid fermentation, the effluent suspended solids as well as effluent COD decreased about 50%.

This research was supported by the Belgian Ministry for Science Policy Programming.

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Accepted for Publication September 23, 1980

lactate and ethanol as intermediates in two-phase anaerobic digestion

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