Conversion of acetate, propionate and butyrate to methane under thermophilic conditions in batch reactors

War. Res. Vol. 25, No. 12, pp. 1509-1513, 1991 Printed in Great Britain. All rights reserved

0043-1354/91 $3.00+0.00 Copyright © 1991 Pergamon Press plc

CONVERSION OF ACETATE, PROPIONATE A N D BUTYRATE TO METHANE U N D E R THERMOPHILIC CONDITIONS IN BATCH REACTORS MUSTAFA (~ZTURK

Department of Environmental Engineering, Ylldlz University, 80750 Yfldtz, Istanbul, Turkey (First received June 1990; accepted in revised form May 1991)

Abstract--These experiments were performed to determine the degradation of VFA (acetate, propionate and butyrate) and the maximum methanogenic activity of granular sludge from the thermophilic anaerobic digestion of pure molasses. The compositions of acetate, propionate and butyrate used as substrate were 25:35:40. The tests were performed at constant temperature (55°C) and pH 7 on two duplicate batch reactors (I and II) running in parallel and were repeated to show the effect of acclimatization. During the first feeding, there was a significant lag phase and after about 23 h incubation the volumes of CH4 gases produced from two reactors were only about 20 and 490 ml, respectively. In this experiment, propionate was converted to acetate only after the initial concentrations of butyrate and acetate had completely degraded. Acetate formed from propionate was immediately converted to methane and carbon dioxide. The maximum methanogenic activities of the first feeding were not high because the natural populations of the propionate-degrading bacteria were low and the sludge adapted itself to the VFAs' substrate very slowly. In a second experiment with the same sludge, the maximum methanogenic activities of the second feedings were about 1.60 times higher than those of the first feedings because of the adaptation of the sludge and increase of populations of the propionate-degrading bacteria. Key words--thermophilic, anaerobic, propionate, batch reactor, methanogenic activity, molasses

INTRODUCTION

The anaerobic digestion process is one of the major biological waste treatment processes in use today. This process has been popular in the waste treatment field, because it has many advantages such as a high treatment efficiency and methane-producing ability (Lin et al., 1986). Volatile acids formed as intermediate compounds during anaerobic treatment are oxidized by floxidation to hydrogen, carbon dioxide and acetate, and the processes are termed dehydrogenation and acetogenesis, respectively. The last stage is methanogenesis (McCarty, 1964; McCarty and Smith, 1986). Methanogenesis involves the reduction of carbon dioxide to methane using hydrogen by relatively fast growing pH-sensitive autrotrophic bacteria. Methanogens also catalyze the reduction of acetate to methane and carbon dioxide (Denac et al., 1988). Thermophilic anaerobic digestion systems offer attractive kinetic advantages in comparison with mesophilic systems. The maximum specific growth rates and activities of thermophilic bacteria are higher than those of mesophilic bacteria, whereas their properties are generally similar to those of mesophilic homologs and the substrate saturation constants are in the same range (Wiegant, 1986), Hydrogen concentration during maximum growth or activities of butyrate converting bacteria is 4.9 times higher than the concentration during maximum

propionate conversion. As a consequence, propionate degradation will be highly inhibited during periods of high activity of the butyrate converting bacteria. Under thermophilic conditions butyrate degrading bacteria are approx. 6 times less sensitive against hydrogen gas than those of propionate. If the hydrogen partial pressure in the reactor is low, acetogens convert propionate and butyrate to acetate, hydrogen and carbon dioxide, but at high partial pressures of hydrogen they are inhibited and propionate and butyrate are not fermented (Table 1). Maximum conversion of propionate and butyrate to methane gas is possible only at low hydrogen partial pressure (Samsoon et al., 1981). Acetate has been described as the least toxic of the volatile acids, while propionate has often been implicated as a major cause of digester failure (Stronach et al., 1986). Propionate conversion appeared to be strongly inhibited, probably because propionate oxidation is thermodynamically rather unfavorable in anaerobic digestion. Methane production from propionate in batch experiments is believed to be slower than that from butyrate or acetate (Gijzen et al., 1988; Hanaki et al., 1987). The methanogenic activity of granular sludges is related to their source and previous feedstocks. During the first feeding the sludge adapts itself to the volatile fattty acid substrate. The activity in the second feeding is higher than the first. This increase may be assumed to be the result of adaption (Field and Sierra, 1989).

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MUSTAFAOZTORK

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Table 1. AG values of some relevant reactionsof 25 and 55°C under the followingconditions;concentrationsof 0.01 M, pH = 7.2, 20% CO2 in the dry biogas and ionic strength I ~ 0.087 M Reaction Substrates

AG(kJ mol- i) Products

25°C

55°C

Equation

Acetic+ H20 CH4+ HCO~-+ H + -26.9 -32.8 4H2+ H + + H C O 3 C H 4 + 3HzO - 127.74 - 22.84 log(pH2) - 114.98 -- 25.15 log(pH2) Acetic+ 4H20 2 H C O 3 + 4H2+ 2H ÷ 100.83 + 22.84 Iog(pH2) 82.17 + 25.15 log(pH2) Propionic+ 3H20 Acetic+ HCOf + 3H2H+ 62.55 + 17.11 Iog(pH:) 48.12 + 18.87log(pH2) Butyric + 2H20 2 Acetic+ 2H~ 32.55 + 11.42log(pH2) 22.84 + 12.55Iog(pH2) Acetic+ propionate + H + + 2H2 Valerate+ 2H20 -48.1 pH2 is the partial pressure of H2. 1/aM of H 2 correspondswith 1270ppm at 25°C and 1370ppm at 55°C in the gas (Wiegant et

1 2 3 4 5 6 al., 1986).

Table 2. Compositionof stock solutions of nutrients and trace elements Nutrient

(g/I)

NH4CI KH2PO4 CaC12.2H20 MgSO4.4H20

170 37 8 9

Trace element (g/I) FeCI3.4HzO COC12-6H20 MnCI2"4H20 CuClz.2HzO ZnCI2 H3BO3

Usually the m e t h a n e p r o d u c t i o n rate during the second feeding is more than 30% higher than during the first feeding (Manual Anaerobic Laboratory, 1989). In this study, specific methanogenic activities o f granular sludge from the thermophilic anaerobic digestion o f pure molasses and conversion o f butyrate, p r o p i o n a t e and acetate fed as substrate to methane were investigated in two batch reactors running in parallel under thermophilic conditions. MATERIALS AND METHODS The experiments were performed in Plexiglas cylindrical reactors with a working volume of 5 dm 3. Reactors were operated at thermophilic conditions (55 + I°C). Two reactors were run in parallel. To each reactor, which contained 15 ml nutrient solution and 2 ml trace elements solution, 2.5g NaHCO 3 buffer was added and they were filled with oxygen-free water. Table 2 shows the composition of stock solutions of nutrients and trace elements. Nitrogen gas was passed through the solutions for 5 min. Granular sludge used in the experiments was originally obtained from an UASB reactor treating pure molasses at thermophilic conditions. The sludge concentration used was 2.5 g organic solids (OS) per liter. The volatile fatty acid (VFA) substrates used throughout the experiments were obtained from a stock solution containing 100:100:100g acetate:propionate:butyrate per kilogram with a pH of 7 neutralized with NaOH solution. The chemical oxygen demand ratio of the VFA stock was 25:35:40 of the total COD for C2, C 3 and C4, respectively. Exact concentrations of VFA used in the first and second experiments were 2.97-3.007g COD per liter in the first reactor and 2.26-2.554 g COD per liter in the second reactor. The VFA standards (C2-C4) were obtained from Merck. The reactors were again flushed with nitrogen gas for another 3 min. The reactor vessels were connected to a gas measurement system. The solutions were continuously stirred for a minimum of 6 s every 3 min during the experiment (Fig. 1). Methane production removed from CO 2 in the gas was measured, daily. Volatile fatty acids (VFA) in the samples taken from each batch reactor were analyzed in a gas chromatograph equipped with a 2 m x 2 mm i.d. column packed with 10% Fluorad FC 431 on supercoat (100-120 mesh). The carrier gas (N2) was saturated with formic acid and flow rate set at 35mlmin -~. The oven

2 2 0.5 0.03 0.05 0.05

(NH4)rMoTO2.4H20 Na2SeO3.5H20 NiCI2"6H20 EDTA HCI 36% Resazurin

0.09 0.1 0.05 1 0.001 0.5

temperature was set at 130°C. The FID detector signal was processed with a SP41 Spectra Physics Integrator. About 163 h later, all the VFA added in the first feeding degraded and the second feedings were made and again methane production was measured. The concentrations of VFA were also analyzed by gas chromatography. RESULTS During the experiments two feedings were made and the temperature and p H o f solutions in the batch reactors remained fai,'y constant at 55°C and 7, respectively. By the end o f 22.66 h methane gas volumes from reactors I and II were 20 ml ( 1 0 m g C H 4 C O D l -l) and 4 9 0 m l (257mg CH4 C O D l-l), respectively. But the methane gas production began to increase sharply after this time [Fig. 2(a)]. During the first experiment, butyrate degraded rapidly, as shown in Figs 3 and 4, but the acetate produced did not immediately convert to m e t h a n e and concentrations o f acetate and valerate continuously increased. After the degradation o f butyrate was completed, acetate started to be converted to methane and carbon dioxide. A b o u t 90.84h later, Stirred SampLe Points

Soda Lime pe~ets

KOH(5%)

TefLon bag Digester

Fig. 1. The stirred batch anaerobic digestion assay set-ups with a Teflon bag for measuring the methane gas production.

Degradation of thermophilic VFAs

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(a)

3

but.yr ate

2--

j/o

'Reactor Z-first feeding

/ , ~ "

;1 E 3

/~L /

O> 0

20

E3 0

I

I

I

t

I

I

I

40

60

80

100

120

140

160 >

(b)

40

~

3

1

yo

0

I 20

/ f J

120

160

Fig. 4. The anaerobic degradation of volatile fatty acids during the first feeding at 55°C in batch reactor II.

~-Reoctor I-second feeding

I 40

I 60

I 80

I 100

I 120

I 140

I 160

Time ( h )

Fig. 2(a and b). The methanogenic activity of pure molasses granular sludge at 55°C during batch anaerobic digestion assays in batch reactors I and II.

degradation of all the acetate was completed and subsequently propionate was converted to acetate. Acetate formed from propionate was immediately converted to methane and carbon dioxide (Figs 3 and 4). The degradation times of acetate, propionate and butyrate for the first feedings are 90.84, 162.92 and 42.5 h, respectively. The maximum methanogenic activity is expressed in milliliters (or g C H 4 C O D ) methane gas production per gVSS of granular sludge in unit time. The methanogenic activity of granular sludge is related to their source and previous feedstocks. The maximum methanogenic activities of the first feeding in reactors I and II were as low as 0.196, 0.201 g CH4 COD/gVSS day, respectively, because the adaptation of sludge itself to VFA substrate and conversion of propionate to acetate took a long time.

During the first feeding the methane obtained was 77 and 79% from VFA based on COD, respectively. The activity period is the time period when the methane gas production rate is at its highest during the feeding. The time period should at least cover about 50% VFA used. The activity period of the first feeding in reactors I and II was calculated to be about 80 h. The methane gas volumes were high for the first days of the second experiment and about 46.6 h later methane production rate continuously decreased as shown in Fig. 2(b). Butyrate and propionate degraded together throughout the second tests and acetate formed from these substrates (C3, C4) was immediately converted to methane and carbon dioxide. Afterwards acetate substrate which was added in the second feeding was converted to methane and carbon dioxide (Figs 5 and 6). The degradation times of acetate, propionate and butyrate for the second feeding were 71, 75 and 27 h, respectively. The maximum methanogenic activities of the second feeding in reactors I and II were 0.285, 0.352 g CH4 COD/gVSS day, respectively. Increase in the

2I

2

ta

/otyrote

/

o

butyrate

-

propionote

o

80

Time ( h ) cond feeding

2

\

*

1

~

n

E

I" 0

40

80

120

160

T i m e (h)

Fig. 3. The anaerobic degradation of volatile fatty acids during the first feeding at 55°C in batch reactor I.

0

40 Time ( h )

..+

80

Fig. 5. The anaerobic degradation of volatile fatty acids during the second feeding at 55°C in batch reactor I.

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MUSTAFAOZTURK

ta

butyrate

Q 0 c.)

>

0

40

80

Time (h)

Fig. 6. The anaerobic degradation of volatile fatty acids during the second feeding at 55°C in batch reactor II. activity in reactors I and II was found to be 45 and 75%, respectively. VFA conversion to methane was found to be 73 and 84% throughout the second feeding. Because sludge adapted itself to VFA substrate and all the VFA degraded within a short time, the activity periods of the second feeding in reactors I and II were as short as about 35 h. DISCUSSION The experimental results of the first feeding indicated that the granular sludge initially adapts to the butyrate substrate. Butyrate is degraded to acetate and hydrogen (Boone, 1989), but its conversion is slow. Acetate formed from butyrate is not immediately converted to methane and carbon dioxide. As shown in Figs 3 and 4, during the first 23 h incubation there is a significant lag phase. After the first 23 h acetate was fermented and hydrogen gas was also consumed by carbon dioxide reducing methanogenic bacteria and as a result methane gas production rate suddenly rose. As in mesophilic digestors, acetate substrate is quantitatively the most important precurser of methane and is the source for about two thirds of methane production in thermophilic digestors (Smith, 1966; Zinder, 1988). After the initial concentrations of butyrate and acetate had completely degraded, propionate substrate was converted by propionate degrading bacteria to acetate only. Acetate formed from propionate was immediately converted to methane and carbon dioxide (Figs 3 and 4). When Figs 5 and 6 are examined during the second feeding, it can be seen that there is no inhibition of propionate degradation. It was observed that propionate and butyrate were simultaneously converted to acetate, hydrogen and carbon dioxide, because of adaptation of the propionate-degrading bacteria. Acetate produced from butyrate and propionate degraded rapidly to methane and carbon dioxide.

The degradation time of propionate in the second feeding is 2.5 times shorter than that of the first feeding. In this case, propionate-degrading bacteria are the most sensitive. Again, according to the results of the first and second feedings, the degradation time of all the VFA in the second feeding is shorter than that of the first feeding. As can be seen from Fig, 2(b), during the second feeding methane gas production rate is fairly high. It is believed that the methanogenic consumption of hydrogen serves to decrease the hydrogen concentrations below the inhibition level. Measurement in continuous cultures have apparently confirmed this phenomena (Kaspar, 1977). Maximum methanogenic activities of the second feeding are 1.45-1.75 times higher than those of the first feeding. This is due to the fact that all the VFA compounds during the second feeding degrade in 63% of the time of those of the first feeding, according to the activity period. This decrease may be considered to be the result of adaptation of the sludge and its propionate-degrading bacteria. As a result of this situation the maximum methanogenic activity should be determined after the second and/or third feeding. The percentage methanogenizations of the volatile fatty acids in the first and second experiments are 77-79 and 73-84%, respectively. This suggests that 15-20% of the VFAs were converted to cells during the first and second feeding. In addition, acetate produced from propionate gas was rapidly converted by methanogens to methane and carbon dioxide, but acetate from butyrate is converted to gases slowly (Figs 3 and 4). This study suggests that natural populations of propionate-degrading bacteria in many methanogenic sludges may be low and that it would be worthwhile to routinely test for their ability to degrade propionate when adapting them to new feedstocks. Acknowledgement--The author wishes to express his sincere gratitude and thanks to the staff of Department of Water Pollution Control, Agricultural University of Wageningen and especially to Professor Dr G. Lettinga for their kind efforts and contribution for the continuation and finalization of this work. REFERENCES

Boone D. (1989) Fermentation reactions of anaerobic digestion. International Course in Anaerobic Wastewater Treatment, IHE, Delft, The Netherlands. Denac M., Miguel A. and Dunn I. J. (1988) Modeling dynamic experiments on the anaerobic degradation of molasses wastewater. Biotechnol. Bioengng 31, 1-10. Field J. and Sierra R. (1989) Biodegradability and toxicity lecture series. Department Water Pollution Control, Agricultural University, Wageningen, The Netherlands. Gijzen H. J., Zwart K. B., Verhagen F. J. M. and Vogels G. D. (1988) High-rate two-phase process for the anaerobic degradation of cellulose employing rumen microorganisms for an efficient acidogenesis. Biotechnol. Bioengng 31, 418-425.

Degradation of thermophilic VFAs Hanaki K., Matsuo K., Nagose M. and Tabato Y. (1987) Evaluation of effectiveness of two-phase anaerobic digestion process degrading complex substrate. Wat. Sci. Technol. 19, 311-322. Kaspar M. F. (1977) Ph.D. thesis, No. 5984, ETH-Zurich, Zurich, Switzerland. Lin C. Y., Sato K., Noike T. and Matsumoto J. (1986) Methanogenic digestion using mixed substrate of acetic, propionic and butyric acids. Wat. Res. 20, 385-394. Manual Anaerobic Laboratory (1989) International Course In Anaerobic Wastewater Treatment, IHE Delft, Agricultural University, Wageningen, The Netherlands. McCarty P. L. (1964) Anaerobic waste treatment fundamentals, Part II. Publ. Wk 123-126. McCarty P. L. and Smith D. P. (1986) Anaerobic wastewater treatment. Envir. Sci. Technol. 20, 1200-1206. Samsoon P. A. L. N. S., Loewenthal R. E., Dold P. L. and Marois G. V. R. (1988) Pelletization in upflow anaerobic

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sludge bed reactors. Proceedings of the 5th International Symposium on Anaerobic Digestion, Bologna, Italy, pp. 55-60. Smith P. H. and Mah R. H. (1966) Kinetics of acetate metabolism during sludge digestion. Appl. Microbiol. 14, 368-371. Stronach S. M., Rudd T. and Lester J. N. (1986) Anaerobic Digestion Processes in Industrial Wastewater Treatment.

Springer, London. Wiegant W. M., Hennink M. and Lettinga G. (1986) Separation of the propionate degradation to improve the efficiency of thermophilic anaerobic treatment of acidified wastewater. Wat. Res. 20, 517-524. Wiegant W. M. (1986) Thermophilic anaerobic wastewater treatment. Anaerobic Treatment a Grown-up Technology, Water Treatment Conference, Amsterdam, The Netherlands.