Stimulation in anaerobic degradation

~,~,arer ResearchVot. 9. pp. 963 to 967. Pergamon Press 1975. Printed in Great Britain.

STIMULATION IN ANAEROBIC DEGRADATION JOHN T. NOVAK Civil Engineering Department. University of Missouri-Columbia. Columbia, Missouri. U.S.A. and MADHUGIRI S. RAMESH Environmental Protection Agency, Evansville, Indiana, U.S.A.

(Received 16 May 1973; in revisedform 20 January 1975)

INTRODUCTION

Anaerobic digestion is usually thought of as being a multi-step process in which complex organics are degraded to short chain acids by facultative bacteria and then to methane and carbon dioxide by methanogenic bacteria. The methane fermentation step has been observed to be the slowest or rate limiting step in this sequence [ l ] and thus kinetic values describing this step should be useful in digester design. Subsequently, enrichment cultures of anaerobic organisms have been subjected to pure feeds of short chain fatty acid, and their carbon removal rates and their energy utilization efficiency or organisms yield determined

E2-41.

It appears that many laboratory studies of biological waste treatment processes are conducted using pure substrates so that data analysis can be simplified. This oversimplification may yield incorrect results due to the ability of microorganisms to metabolize substrates faster in the presence of stimulatory organics. This study was designed to delineate the nature of stimulation of acetic acid degradation by the addition of organic materials and to ascertain which organics and what degree of stimulation might be expected.

METHOD OF STUDY While such studies have providedvaluable informaAn enrichment culture of methane bacteria acclimated tion which can be incorporated into the design of to sodium acetate was developed from anaerobic digester these bacterial mediated treatment units [5], certain sludge taken from the activated sludge treatment plant, field units have been shown to be more efficient in Columbia, Missouri. A 6.2 liter plexiglass laboratory digesremoving wastes than any of the laboratory units ter was intermittently fed sodium acetate along with an receiving pure volatile acid feeds. It appears that un- inorganic nutrient solution on a fill and draw basis for 2 months to ensure a complete washout of all but those der certain undefined conditions, the anaerobic bac- organisms acclimated to acetate. The composition of the teria can degrade organics at rates faster than that inorganic nutrient solution was identical to that used by observed in laboratory units which are fed a sole car- Lawrence and McCarty 1"2]. Feeding was conducted on at least a once-a-week basis and about 0.25 of the contents bon source. of the unit were exchanged. Attempts to stimulate the biological activity of Since methane bacteria are capable of degrading only anaerobic digestion units have centered around pro- a few setlect short chain fatty acids 1"8], any other organic viding some unknown growth factor, usually a heavy substrate would remain undegraded in the presence of the metal or vitamins. Others have advocated pre-packed enrichment culture. If growth stimulation does occur, it enzymes, assuming that these will have the same effect would probably not be evidenced by removal of the nonfatty acid organic, but could be most easily seen by comas maintaining a high active organism population. parison of the fatty acid degradation rate or the organism Some success has been achieved with these attempts, yield to units which contain no stimulating organics. The chiefly the delineation of required heavy metals by initial phase of this study was to determine the degree Speece and McCarty [6]. Other attempts have pro- of stimulation which would occur from the addition of growth organic intermediates. The second phase of the vided erratic results, at times providing surges in gas study consisted of organism yield measurements in the production but generally failing to generate a consis- absence and presence of organic stimulants. tently higher rate. One method of stimulation which Batch studies were prepared by placing 200 ml of the has been successful although it has not found wide- enrichment culture into 500 ml Erlenmeyer flask containspread use in the field has been to return a portion ing dry sodium acetate and organic materials selected for study. The flask shown in Fig i was equipped with a rubber of the dry digested sludge material. McCarty and balloon on one end to pressurize the vessel and an outlet Vath [7] found acetic and butyric acid degradation tube and clamp to provide sampling without exposing the rates could be enhanced by the addition of dried contents of the flask to the atmosphere. The flask was filled supernatant solids. The stimulatory matter in these to 400 ml with tap water and flushed with nitrogen gas solids was apparently of organic origin since addi- and then pressurized. A magnetic stirrer continuously mixed the contents of the flask. The flask was maintained tions of the ash of this material caused no increase in a constant temperature incubator at 370C, which is the in gas production. general area of digester operation and is the optimal :~63

964

J o H x T. NOVAK a n d MADHLGIRI S. RAHESH

growth temperature for mesophylic digester methane bacteria. All results in the initial phase were evaluated by comparison to acetate degradation rates in the absence of any additional organic substrate. Acetate concentrations were measured as total volatile acids by the column chromatographical technique in Standard Methods [9]. The concentration of total organic matter was measured as soluble organic carbon on a Beckman Total Carbon Analyzer. The contribution of acetate to the total organic carbon was calculated from the volatile acid measurements• In this manner, acetate carbon, total carbon and the carbon concentration of the added stimulatory organics were determined. Growth organics selected for this study consisted of either known biological growth intermediates or a mixture of undefined cellular materials. Amino acids were selected because they comprise about 50% of the dry organic solids as proteins [10]. Nucleic acids, nucleosides and ribose comprise some of the components of RNA and DNA, so were also considered to be potential stimulants. Components of RNA and DNA used in this study were thymine, uridine, ribose, adenylic acid and guanylic acid. Cellular materials were obtained by lysing both anaerobic and aerobic bacteria. The two groups of amino acids used to investigate the stimulation phenomenon were as follows. Group (A) glycine, serine, theonine, aspartic acid, and glutamic acid. Group (B) tryptophan, phcnylalanine, lysine monohydrochloride, valine, and glutamic acid. Cellular organic materials were obtained by two methods. Both aerobic organisms (activated sludge) and anaerobic bacteria (enrichment culture) were autoclaved for 30min, filtered through a glass fiber and then dried. Anaerobic bacterial organics were also obtained by bubbling pure oxygen gas through a portion of the enrichment culture to cause cell lysis, filtered and dried. These dried materials were then fed to the batch study units.

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Fig. 2. Increased acetic acid degradation resulting from additions of amino acids. For the organism yield experiments a stock solution containing equal weights of valine, thrconine, lysinc and monohydrochloride, tryptophan, meLhionine, phenyla. lanine and glutamic acid was prepared. Organism biomass was measured as volatile suspended solids f'¢~dS):as prescribed by Standard Methods [9"]. The acetate con. centrations were again determined by column chromatography. • RESULTS The'stimulatory effect of amino acid additions are shown in Figs, 2-4. Figure 2 is a plot of accumulated acetic acid degradation for the amino acid stimulated units and the control. Figure 2 shows that increased acetic acid degradation occurs in the presence of amino acids for the duration of the experin~nt.

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EXPERIMENTAL APPARATUS Fig. 1. Experimental batch reactor.

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Fig. 3. Effect of varying concentrations of Group (A) amino acids on acetic acid degradation.

965

Stimulation in anaerobic degradation 200 E

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Fig. 4. Effect of varying concentrations of Group (B) amino acids on acetic acid degradation.

In Figs. 3 and 4, the effect of several concentrations of amino acids is shown. For the Group (A) amino acids, essentially equal stimulation was observed for both the 50 and 100mgl - t concentrations. At 150mgl-' inhibition occurred. These results are in agreement with the findings of Milholits and Malina [11] who observed that glycine had a stimulatory effect on gas production in laboratory anaerobic digester units at a concentration of 1.08 m moles !- t, but at a concentration of 4.0m moles 1- [ inhibition occurred. Since the Group (A) amino acids contained glycine, the inhibition could have resulted from this amino acid. The Group (B) aminGacids.showed an increasing rate of acetic acid degradation as the concentration of amino acids increased. In Fig. 5, changes in soluble organic carbon with time are plotted for stimulation by 100 mg 1-t concentration of the Group (B) amino acids in order to determine the fate of the stimulatory amino acids. Acetic acid carbon values were calculated from actual acetic measurements. The difference between the measured total organic carbon and calculated acetic acid

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Fig. 6. Effect of nucleic acid components on acetic acid degradation. carbon were assumed to belong to the amino acids. As can be seen from the upper plot in Fig. 5 the amino acid carbon level slowly decreased. The effect of thymine, uridine, ribose, adenylic acid, and guanylic acid additions on acetic acid uptake at a concentration of 50 rag 1- t is shown in Fig. 6. Both thymine, a pyrimidine, and uridine, a nuclcoside increased the rate of acetic acid degradation over that 16001

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Fig. 7. Stimulation of acetic acid degradation by additions of bacterial lysate.

966

JOHN T. NO',.~,Kand ~IADHLGIR[S. RAYmSH

Table h Organic carbon fractions during degradation --stimulation by thymine and uridine TIME (4eys)

TOTAL SOLUBLE ORGANIC CARS0~

ACETIC ACID CARBON (mr/I) 544 440 352 176

DIFFERENCE (mq/t)

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DISCUSSION

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of the control. Adenylic acid and guanylic acid, both purines, inhibited the acetic acid degradation. The carbon values for thymine and uridine are shown in Table 1. These results show that the non-acetate carbon fraction is nearly constant, indicating that increased acetic acid degradation rates are not due to increased concentrations of degradable substrate but are the result of stimulation.

In Fig. 7, data obtained for acetate degradation in the presence of biological materials derived from

anaerobic bacteria (enrichment culture) is shown. In 8 days, autoclaved anaerobic bacterial derivatives increased the amount of acetic acid degraded by 52~.

This degree of stimulation was the greatest observed during any of the batch experiments. Organics derived by bubbling oxygen gas through the enrichment

culture also produced an increase in acetate degradation. A 36~ increase in acetate degradation occurred after 8 days. In Fig. 8, acetic acid degradation and changes in organism biomass in batch cultures stimulated by organics derived by autoclaving the enrichment culture organisms are compared to cultures where no stimulants were added. Organism yields are shown

in Table 2 for the enrichment cultures of methane bacteria in the presence and absence of organic stimu-

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lants. Amino acids increase the yield from ().t28 to 1.35mg VSSmg acetic acid while the cellular organics increase the ~ields by about 40°,,.

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Acetic acid is degraded more rapidly anaerobically in the presence of organics which are required as essential celt constituents including amino acids and nucleic acids. In Figs. 3. 4 and 6. the increasing amounts of acetate degradation in the presence of organic stimulants are plotted with time. The constantly increasing amount of acetate degradation with time in these plots indicates that the effect of stimulatory organics is one of increasing the growth rate of the organisms. Since no extra degradation energy source is provided the effect may be one of direct assimilation and use of these growth intermediates without the energy expenditure required to manufacture these organics from inorganic materials. From Fig. 3 it can be surmized that there is an

optimum concentration of the Group (A) amino acids for maximum stimulation. Increasing the concentration above this would inhibit the growth and result in lower degradation rates. This observation is in agreement with the findings of Milholits and Malina 1-11]. Glycine, one of the ingredients of Group (A) amino acids is observed to retard growth when present in high concentrations. Increases in the concentration of Group (B) amino acids always resulted in an increase in acetic acid degradation. It appears that saturation or maximum stimulation would occur at concentrations of Group (B) amino acids in excess of 150mgl -t. Stimulation by the nucleic acids components are comparable to that of the amino acid mixtures. In 7 days, both thymine and uridine at a concentration of 50 mg 1- t increased the acetic acid degradation by 13 and 17)o respectively. Adenytic acid, and guanylic acid at 5 0 m g l - i had an inhibitory effect on the organism growth. Natural biological stimulants derived by lysing the methane bacteria provide the greatest degree of stimulation. Since autoclaving and drying were used in obtaining these natural biological stimulants,

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Table 2. Increases in organism yield in the presence of stimulatory organics rIME idays|

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LYSED AN A li[NOelC CELLULAR MATTER ADDED (40rag C/I )

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ACETIC ACID OEGRAO[D mg/I

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Stimulation in anaerobic degradation enzyme denaturation would have occurred and therefore the possibility of stimulation by the addition of enzymes is not likely. These natural biological stimulants are probably similar to those found in digester supernatant and would explain the stimulation previously observed resulting from additions of these materials to anaerobic cultured receiving an acetate feed. Other dissolved organics such as vacuum filter liquor or the liquor from sludge heat treating processes would probably provide an equal degree of biostimulation if recycled. In the absence of organic growth stimulants, the average microorganism yield coefficient is 0.128mg VSS m g - t acetic acid. This is equivalent to a yield coefficient of 0.137 when expressed in mg VSS mg-t COD units. When 70mg - t amino acids were present the yield value increased to 0.139 mg VSS-t mg-t acetic acid. In the presence of biological stimulants derived by lysing the methane bacteria, the yield was 0.180 mg VSS mg-~ acetic acid. The higher yield coefficients obtained by addition of growth organics and the increasing difference in the biomass plotted in Fig. 8 suggests that the increased acetic acid degradation achieved in the presence of growth intermediates are the result of an increase in the biomass and not due to any other phenomena such as an increase in enzyme activity. In general, these results indicate that mixed or complex substrates would probably be degraded at faster rates than simple or pure substratesdue to the mutual stimulation resulting from the biological growth intermediates. Kinetic values for the anaerobic degradation of sole substrates may be considered to be the slowest or limiting case encountered in uninhibited digester operations. Sewage sludge degradation rates should be faster than those observed to occur in laboratory studies using pure feeds depending upon the feed concentration of the sludge. CONCLUSIONS As a result of this study the following conclusions seem to be justified. I. Methogenic bacteria are able to degrade acetic acid more rapidly in the presence of growth intermediates that are required as microorganism cellular components.

967

2. A mixture of amino acids at a concentration of 150mg 1-t increased the acetic acid degradation by 17°o. 3. Organics derived by lysing methane bacteria increased the acetic acid degradation by 52}~. 4. In complex substrate systems, individual waste constituents should be degraded more rapidly than as sole substrates. Therefore, kinetic values obtained for pure substrates may not be applicable to mixed substrates systems. Acknowledgement--This project was supported in part by funds from the Office of Water Resources Research. U.S. Department of the Interior, Project N o. A-039-MO. REFERENCES

[1] McCarty P. L. (1964) Anaerobic waste treatment fundamentals: part l, chemistry and microbiology, Public Works, Sept. [2] Lawrence A. W. and McCarty P. L. (1969) Kinetics of methane fermentation in anaerobic treatment. J. War. Pollut. Control Fed. 41, Rl. [3] Novak J. T. and Carlson D. A. 0970) The kinetic of anaerobic long chain fatty acid degradation. J. War. Pollut. Control Fed. 42, 1932. [4] Schulze K. L. and Raju B. N. (1958) Studies on sludge digestion and methane fermentation--If: Methane fermentation of organic acids. Sewage ind. Wastes 30, 164. [5] Lawrence A. W. and McCarty P. L. (1970) Unified basis for biological treatment design and operation. J. San. Engng Dir. Am. Soc. cir. Engrs 96. No. SA3, 757. [6] Speece R. E. and McCarty P. L. (1964) Nutrient requirements and biological solids accumulation in anaerobic digestion. Advances in Water Pollution Research. Proc. 1st Int. Conf. Wat. Pollut. Res. Pergamon Press, New York. [7] McCarty P. L. and Vath C. A. (1963) Volatile acid digestion at high loading rates. Int. J. Air War. Pollut. 6, 65. 18] Cookson J. T. and Burbank N. C. (1965) Isolation and identification of anaerobic and facultative bacteria present in the digestion process. J. War. Pollut. Control Fed. 37, 822. [9] Standard Methods for the Examination of Water and Wastewater (1967), 12th edn. Am. Public Health Assn.. New York. [10] Stanier R. Y., DuodoroffM. and Adeiberg E. A. The Microbial World. Prentice-Hall, Englewood, New Jersey. [ll] Milholits E. M. and Malina J. F., Jr. 0968) Effects of amino acids on anaerobic digestion. J. War. Pollut. Control Fed. 40, R42.