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The effects of chloroform toxicity on methane fermentation
War. Res. Vol. 20, No. 10, pp. 1273-1279, 1986 Printed in Great Britain.All fights reserved
0043-1354/86$3.00+0.00 Copyright © 1986PergamonJournals Ltd
THE EFFECTS OF CHLOROFORM TOXICITY ON METHANE FERMENTATION JOSEPH YANG and R. E. SPEECE Environmental Studies Institute, Drexel University, Philadelphia, PA 19104, U.S.A. (Received October 1985) Abstract--Tbe methanogenic phase of anaerobic digestion is often considered to be the most sensitivestep in the overall process. Chloroform, a model industrial toxicant was assayed with methane fermentation, acetate enrichment cultures. Commonly, microbial toxicity response studies have observed the toxicity response over a relatively short time span, on the order of a few hours or days. In addition, cessation of observed cell function has often been assumed to be equated with death of the cells. The purpose of this study was to determine the toxicity response under various conditions over prolonged periods, on the order of months, with major emphasis on the recovery pattern. The following parameters were observed: concentration of toxicant, solids retention time, biomass concentration, toxicant exposure time, cell age, toxicant administration pattern and temperature. The methane fermentation culture was able to acclimate to the presence of chloroform while fermenting acetate to methane. Inhibition of unacclimated cultures was noted at 0.5 mg I-l, but with acclimation 15 mg I-l could be tolerated. There was a clear correlation between dose and inhibition. Recovery from inhibitionwas a function of SRT and biomass concentration. Younger cells were more resistant than older cells. The duration of chloroform exposure was directly related to the degree of residual inhibition. Reduced temperature delayed recovery of gas production from slug doses of chloroform. Radio tagged chloroform studies indicated that recovery of methane production occurred due to acclimation of the methanogens to the presence of chloroform and not from the disappearance of chloroform by microbial degradation. Key words--chloroform, toxicity, methane, fermentation, anaerobic digestion
INTRODUCTION The anaerobic digestion process for waste treatment is a mixed culture system which converts organic wastes to methane gas under anaerobic conditions. Normally, methane fermentation occurs satisfactorily in anaerobic sludge digestion and has an excellent potential for the treatment of warm industrial wastewaters. However, a number of features in regard to its response to toxicity have to be identified in order to insure reliable process performance. Commonly, microbial toxicity response studies have observed the toxicity response over a relatively short time span, on the order of a few hours or days. In addition, cessation of the observed cell function has often been assumed to be equated to death of the cells. The purpose of this study was to determine the response of the acetate conversion to methane step of anaerobic digestion under various toxicity conditions over prolonged periods, on the order of months, with major emphasis on the recovery pattern. The parameters which affect the toxicity of foreign substances are not only related to the kind of toxicant substances, but are also related to other factors. Some factors which can affect toxicity response are: concentration of toxicant, solids retention time (SRT), biomass concentration, toxicant exposure time, cell age, toxicant administration pattern, and temperature. These factors were assayed in this study.
The methanogenic phase is often considered to be the most sensitive step in the anaerobic digestion process. Therefore, it was determined to explore the response of acetate utilizing methanogens to a sample toxicant, chloroform, which is widely used industrially and is found in industrial waste effluents. Chloroform is very toxic to methane fermentation. Stickley (1970) reported 0.01 mg 1-I or more of chloroform in the sewage is likely to have an adverse effect on sludge digestion. Swanwick and Foulkes (1971) found chloroform to be most toxic to the anaerobic digestion of sewage sludge when compared with five other chlorinated hydrocarbon solvents including carbon tetrachloride and l,l,l-trichloroethane. Bauchop (1967) and Thiel (1969) used chloroform as a special inhibitor in studying the inhibition of methanogenesis owing to its being an analogue of methane. Swanwick and Foulkes (1971) concentrated on studying chloroform in anaerobic digestion due to its toxicity and the frequency with which it had caused difficulties in anaerobic digestors in Britain.
PROGRAM OF STUDY Seven sets of experiments were designed to evaluate the response of acetate methane fermentation to chloroform toxicity. These experiments were designed primarily to elucidate the role which the
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1274
JOSEPH YANG and R. E. SPEECE
following process design and control parameters play in toxicity response: (!) (2) (3) (4) (5) (6) (7)
Toxicant concentration SRT Biomass concentration Toxicant exposure time Cell age Toxicant administration pattern Temperature.
In this section, only a general outline of the program of study for each experiment is given. The re!eyant procedures for each experimental study are presented in each respective subsection of the results section to minimize redundancy.
Toxicity response according to toxicant concentration The response of methane production in semicontinuously fed methanogenic cultures to one-time, slug doses of chloroform was determined. Chloroform concentration was the variable parameter. With 50 day S R T systems, the microorganisms were exposed to 0.5, l, 2, and 2.5 mg l -t of chloroform. With 25 day S R T systems 0.5, l, and 2.5 m g l -l of chloroform concentration was assayed. With 12.5 day S R T systems, 0.25, 0.5, and 2.5 m g l -~ were assayed.
Toxicity response according to S R T In these studies, the experiment was similar to Item 1 above. However, S R T was the variable parameter. Chloroform concentrations tested at three S R T levels were 1 and 2 . 5 m g l -l.
Toxicity response according to biomass concentration Three different biomass concentrations were prepared in serum bottles and exposed to a range of concentrations of toxicant. One set had a biornass concentration in the serum bottles which was the same biomass concentration (925 mg l -t) as in the 50 day S R T inoculum culture. A second set had double this concentration, while the third set contained half of the biomass concentration of the 50 day S R T inoculum culture.
Toxicity response according to toxicant exposure time The cultures were exposed to a given chloroform concentration for the desired exposure time. The cells were then centrifuged, and the supernatant was withdrawn and replaced by unadulterated supernatant. Subsequently the gas production response was noted.
Toxicity response according to cell age The age of cells is reflected in the SRT. However, S R T reflects not only age of cell, but also biomass concentration. With a different SRT, both age and biomass concentration in reactors are different when loading rate was kept constant at 1000 mg l-l-day of acetate. Therefore, in these toxicity studies the effects of cell age on toxicity response were separated from the effects of biomass concentration. When assaying
the effect of cell age on toxicity, the reactors were adjusted to contain equal biomass concentrations with only cell age as a variable. They were operated as batch reactors subsequently.
Toxicity response according to toxicant administration patterns The chloroform was administered in two feeding patterns. Continuous dose of chloroform in the feed resulting in a gradual build-up in the culture. Slug dose to the culture resulting in first order washout.
Toxicity response according to temperature Two incubation temperatures were assayed, 25 and 35°C, at 50 day SRT. A slug dose of 1 m g l -~ of chloroform was added to both. MATERIALS AND METHODS
Inoculum source and feeding solution Methanogenic cultures enriched on acetate as the sole organic-carbon source (exclusive of 10mgl -~ of cysteine) were used in this study. A 4001. stainless steel CSTR inoculum digester with semi-continuous feeding and wasting, was maintained at a 50 day hydraulic and solids retention time and fed 1 g l-l-day of acetic acid. Glass carboys were used to culture the 25 and 15 day SRT inocula. All three inocula sources were operated in excess of three SRT before being used in this study. A defined nutrient salt solution, described elsewhere was used to maintain the inocula (Yang and Speece, 1986). Serum bottle technique The serum bottle technique as developed by Miller and Wolin (1974) and modified by Owen et al. (1979) was further modified for this study. The 50ml culture volume was anaerobically transferred into oxygen-free bottles of 125 ml volume. Once seeded, the serum bottles were operated in either batch or semi-continuous mode. After preliminary replicate experiments established the close replicability of results (the range of gas production variation for six replicates averaged < 10% with a maximum of 20%) all assays were run without further replication. The serum bottles were shaken once daily and incubated at 35°C except for the one assay indicated at 25°C. In a semi-continuous study, the serum bottles were maintained at a given SRT, the same SRT as the inoculum digesters, by daily withdrawal of the requisite volume of mixed contents and replacement with the nutrient salt solution using hypodermic syringes. Gas production was measured daily. The acetic acid concentration was stoichiometrically restored daily to 1000 mg 1-~ by adding glacial acetic acid with a microliter syringe in proportion to the volume of methane produced the previous day. In a batch study, no wasting of mixed contents or feeding of nutrient salt solution was conducted. Only gas production was measured and the acetic acid concentration was stoichiometrically restored to 1000 mg 1-I each day based on the volume of methane produced. ANALYTICAL PROCEDURES
Gas measurement Gas production in the serum bottles was measured by displacement of an acidified salt solution in a 100 ml graduated cylinder connected to a hypodermic needle which pierced the serum cap. Volatile suspended solids The biological solids were measured as volatile suspended solids (VSS). The analysis was performed according to
Effect of chloroform toxicity on methane fermentation Standard Methods (APHA, 1975). Glass fiber filters were put in a 550°C oven for 15 ruin before filtration to insure no volatile matter was present in the filters. Chloroform measurement The chloroform concentration in the cultures was measured by gas chromatography. A Shimadzu C_~-6 AM gas chromatograph with a glass column 3 ft x 3 mm i.d. containing packing material OV- 101 on 80/100 Supelcoport supplied by Supelco Inc., was used. Helium was employed as the carrier gas. The flow of carrier gas was 40 ml rain- 1. The injection temperature was 150°C and the column temperature was 85°C. Radioactive tagged chloroform analysis The radioactively tagged chloroform, [ ~4q was purchased from New England Nuclear, Boston, Mass. One ml of radioactively tagged chloroform sample was diluted into 10ml of liquid scintillation cocktail in plastic vials, then vigorously shaken. Counting was done with a Beckman LS-100C Liquid Scintillation System. The time for counting was set at 50 rain. The efficiency error was set at 1%. The samples were tagged at the level of 1000cpm. The background was 60cpm. A 48% counting efficiency was documented. RESULTS AND DISCUSSION Toxicant concentration Three SRT (50, 25, and 12.5 days) ranges were maintained in the serum bottles with respective VSS levels of about 925, 780, and 515 mg 1-l. A specified volume of mixed contents was wasted (1, 2, and 4 ml per day) and the same volume of nutrient solution was fed each day to maintain each given SRT. Gas production was measured daily. Several days of gas production were recorded prior to injection of the indicated slug doses of chloroform to establish a base line. The partitioning of chloroform between the liquid and gas phases was experimentally determined to be 3.1 at 35°C with 50 ml of liquid volume in a 125 ml serum bottle. The theoretically calculated partition coefficient was 2.9 under these conditions. The equations used for this calculation are shown in the Appendix. For our conditions, 68% of the initial injection of chloroform remained in the liquid phase after equilibration.
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The tendency of chloroform to adsorb/absorb to the biomass was evaluated by injecting radio-tagged chloroform into cultures containing 0 (initially centrifuged supernatant), 1, 2, and 4 times the biomass concentration of the 50 day SRT inocula (925 mg 1- ]) and allowing 6 days of equilibration. Then the cultures were centrifuged and the radioactivity in the supernatant determined. The control (initially centrifuged supernatant) was 4300 cpm. The 1 x concentration of biomass was 3800 cpm (88%), while the 2 x and 4 x concentrations of biomass were 3500 cpm (81%). In view of the fact that the counting precision was 200 cpm, these results indicate that adsorption/absorption of chloroform by the biomass reduced the liquid phase chloroform by 12% in 50 day SRT cultures. Figure 1 shows the results of chloroform toxicity on 50 days SRT methanogens. The chloroform concentrations shown in this study are the concentration initially injected into each system. Chloroform volatility caused it to be stripped by the gas production. The concentration of chloroform therefore decreased with time by gas stripping and liquid washout. Figure 2 shows how the calculated chloroform concentration would change by stripping due to gas production and liquid washout after 2 mg 1- l chloroform was injected into a 50 day SRT serum bottle. This calculated curve was verified by radioactively tagged chloroform experiments. A small difference in chloroform concentration can have a disproportionately large effect on recovery of methane production if the gas stripping ceases due to cessation of methane production as shown in Fig. 1 comparing a chloroform dose of 2 and 2.5 mg 1- i. A prolonged period of zero gas production would tend to lead one to the conclusion that the methanogens were "dead". However, the rapid gas production recovery from 2.5mgl -j would suggest the system biomass remained viable during this pro-
~, 2 m g / t chloroform injected into the 50 day SRT Serum Bottle -.. 2 0
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1276
Joseph YANO and R. E. SPF.ECE 15mg/t Chloroform •
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Fig. 5. Effect of biomass conch on response to c h l o r o f o r m - 2mgl-L
longed period of zero gas production with metabolism blocked until detoxification and/or acclimation to the chloroform occurred. With the hypothesis that negligible bidtransformation was occurring, the chloroform concentration in a 50 day SRT digester was calculated for a 20 day period based upon the actual daily gas production and liquid wastage after a slug dose of 1.5 mg 1-l. During this same period carbon 14 tagged chloroform was spiked into the reactor and the radioactivity in the effluent samples was counted daily. As shown in Fig. 3, there is close agreement between the actual chloroform concentration based upon radioactivity in the effluent and that calculated on the assumption that biotransformation is insignificant and only gas stripping and liquid washout account for chloroform loss. Bouwer and McCarty (1983) demonstrated anaerobic metabolism of chloroform at 0.1 mg 1- ~ concentrations, which is 1/15 of the concentrations used in this study. Radio tagged chloroform studies with concentrated biomass, referred to previously showed the same supernatant radioactivity after 6 d~ys with 2 x and 4 x biomass Concentrations. If bidtransformation of chloroform was occurring, the radioactivity in the supernatant of the 4 x biomass concentration should have been less than in the 2 x .
12.5 day SRT in Fig. 4. Figure 4 shows that the 50 day SRT gas production recovered to normal faster than the 25 or 12.5 day SRT system. Also, the methanogens at a 50 day SRT could recover from 2 . 5 m g l -~ of chloroform from which the methanogens at 12.5 and 25 day SRT did not recover. Generally, it is considered that the greatest SRT provides the greatest process stability in the presence of toxic shocks to the biomass. The chloroform toxicity responses for 50, 25, and 12.5 day SRT semi-continuous systems are summarized in Table 1. In this case, the advantage of extended SRT in minimizing chloroform inhibition was demonstrated by increased gas production recovery rates at 50 days when compared with 25 and 12.5 day SRT, except for the case of 0.5 mg 1- ~ where all recovery times were equal. There are three factors which express the differences among SRT. One factor is biomass. In this study, the biomass at 50, 25, and 12.5 day SRT digesters was 925, 780 and 515mgl -~ respectively. The second factor is age of cell. The age of organisms in a digester is reflected in SRT. The third factor is the washout of toxicant and biomass. The concentration of toxicant is washed out faster in low SRT system. Therefore, this would tend to reduce inhibition.
SRT
Biomass concentration
In this study, gas production cross plots for 1 mg 1- l of chloroform are plotted for 50, 25, and 1 mglI Chloroform
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Effect of chloroform toxicity on methane fermentation fuged at 4000 rpm for 15 min and 50 ml of cell free supernatant fluid was withdrawn. Thus, these serum bottles then had double the biomass concentration of the standard 50 day SRT inoculum culture. When the serum bottles were seeded with 25 ml of methanogenic culture from the 50 day SRT inoculum culture and injected with another 25 mi of cell-free supernatant fluid, these serum bottles were considered to be at half the biomass concentration of the standard 50 day SRT inoculum culture. After preparing the serum bottles containing the desired biomass concentrations, 1000 mg 1-~ acetic acid was injected into each bottle. Then gas production was measured daily. The acetic acid level was stoichiometrically restored to 1000 mg I-l daily. Subsequently, various concentrations of chloroform were injected into each of the systems containing different biomass concentrations. During this experiment, no withdrawal of mixed contents or replenishment of nutrient solution was practiced in order to attempt to maintain the different relative biomass concentrations in the serum bottles. Three different biomass concentrations were evaluated in this phase. The results are shown in Fig. 5. When 2 mg I-l of chloroform was administered to the systems, the gas production in the system containing the highest biomass concentration recovered sooner than that of the lower biomass concentration systems. Toxicant exposure time
Serum bottles were seeded from the 50 day SRT inoculum culture and were injected with various concentrations of chloroform. After the desired exposure time (1 h, 1 day, etc.), the serum bottles were placed directly into a centrifuge and centrifuged at 4000rpm for 15min. They were then carefully inverted and the supernatant liquid phase was completely removed using a syringe. (In this study, toxicant exposure time refers to liquid phase only and ignores the 12% of toxicant absorbed on the biomass.) A 67/33% mixture of N2/CO~ was introduced while the supernatant liquid was withdrawn. Then, unadulterated, centrifuged supernatant fluid from the inoculum culture was injected into the serum bottle to replace the unadulterated supernatant fluid withdrawn. Daily gas measurement and stoichiometric acetate compensation were then continued as described before. During the experimental period, no mixed culture contents were wasted and no replenishment of nutrient solution was practiced. This phase of study was designed to elucidate the effect of toxicant exposure time on gas production recovery. Figure 6 shows the 1 h and 24 h exposure time responses to 50 and 200 mg 1-] of chloroform. When methanogens were exposed to 50mg 1-1 of chloroform the recovery of methane fermentation after 1 h chloroform exposure was faster than that for 24 h chloroform exposure.
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Chloroform 1 hr or 24 hrs Exposure
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Fig. 6. Effect of exposure time on response to chloroform. When the chloroform was removed from the liquid phase by replacing with unadulterated supernatant fluid, the gas production recovered after the lag periods shown. This indicates chloroform was bacteriostatic, but not a bactericidal and bacteriolytic agent in the concentration ranges and exposure times assayed. These results indicate the potential importance of rapidly separating the toxicant contained in the liquid phase from the biomass which is one advantage of the "plug-flow" anaerobic filter. Yang et al. 0980) found that continuous feeding of unadulterated nutrient salt solution into anaerobic filters subsequently after a one-day slug dose of 300 mg l - mof chloroform allowed the gas production to recover within l0 days. Although the dilution capability of a CSTR (completely stirred tank reactor) is considered to be a major advantage, it is now clear that, in some cases, not toxicant dilution, but either rapid removal of toxicity from contact with the biomass or toxicant removal, ensures more rapid recovery than does initial dilution followed by slow washout of the toxicant from a CSTR. Effect o f previous exposure on response
Figure 7 shows the relative response of the methanogens after previous exposure to various chloroform concentrations. Separate serum bottles were spiked with 0.5, 1.5, and 2 . 0 m g l -m of chloroform. These systems were then operated at 50 day SRT with 2.5 m g l l Chloroform
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1278
JOSEPh YAr~o and R. E. SPEECE
daily restoration of the volatile acids concentration to 1000mg1-1 by stoichiometric supplementation of acetic acid in proportion to the previous days' methane production. After all of the systems had recovered to normal gas production, 2.5 mg1-1 of chloroform was reinjected into all cultures, including a control which had not previously been exposed to chloroform. As shown in Fig. 7, the system with the most protracted recovery time was the unacclimated control. The culture previously acclimated to 0.5 mg 1-1 chloroform had the second most protracted recovery. The culture previously exposed to 1.5 mg 1-~ chloroform demonstrated the shortest recovery period. This indicates that previous exposure of the methanogens to an "optimum" chloroform concentration facilitates acclimation which is manifested in reduced inhibition from subsequent exposures to chloroform.
Ce//age Cell-free supernatant fluid was used to dilute the biomass concentration in the 50 day SRT serum bottles down to the same VSS concentration as that found in the 12.5 day SRT serum bottles. The VSS concentration in the 50 day SRT inoculum culture was about 925 mg 1-1 and this was diluted down to 515 mg 1-l which was the biomass concentration in the 12.5 day SRT systems. Then the toxicity response pattern was evaluated in batch systems with no wasting of the mixed contents but stoichiometric restoration of acetate daily to 1000 mg 1-1. The results are shown in Fig. 8. With 1.5mg1-1 chloroform injected into the methanogenic cultures, the gas production in the 12.5 day $RT system took about 9 days to fully recover to the background rate, but the same biomass concentration of 50 day SRT methanogens had only recovered to 50% of the background rate after 17 days. Therefore, the younger cells of 12.5 day SRT were more robust and less adversely affected by chloroform than were equal biomass concentrations of 50 day SRT cells. Baresi et aL (1978) found H: and formate inhibition of methane fermentation from acetate were also related to the age of the enrichment culture.
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Fig. 9. Effect of gradual build-up of chloroform on response---50 day SRT. Toxicant administration pattern
In this phase of the investigation the chloroform toxicity response was compared for slug doses vs gradual administration in the feed. The gradual administration of toxicant simulates a waste stream of constant toxicant concentration being fed to a CSTR. With gradual administration of chloroform concentrations, the 50 day SRT culture could tolerate about 15 mg !-1 chloroform in the feed as shown in Fig. 9. However, with the 25 day SRT culture, only 5 mg !- l of chloroform could be tolerated in the feed as shown in Fig. 10. With the 12.5 day SRT culture, Ci°r°f°rm l e° c~o
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when the biomass was separated from the supernatant fluid containing chloroform. (6) Previous exposure to chloroform reduced inhibition due to subsequent reinjection of chloroform. (7) At equal biomass concentrations, younger cultures proved more robust and resistant to chloroform than older cultures. (8) Reduced temperature (25°C) significantly delayed recovery of gas production from slug doses of chloroform when compared to the normal incubation temperature (35°C).
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Fig. 12. Effect of incubation temperature on toxicity response to chloroform--1 mgl -t. even 1 mg 1-1 of chloroform in the feed caused eventual failure of gas production as shown in Fig. 11. By comparison, a slug dose of only 2.5mgl -~ was devastating to all systems. Temperature
Toxicity response characteristics were investigated at two different incubation temperatures, i.e. 25 and 35°C. Serum bottles were maintained at 50 day HRT and SRT by daily wasting of 1 ml of mixed contents and replacement with 1 ml of nutrient solution. Acetate was stoichiometrically compensated daily to 1000mgl -l according to methane production. The systems were operated for 3 days prior to injection of chloroform in order to establish the background methane production rate. At this point the same concentration of chloroform was injected into bottles which were incubated at 25 and 35°C. The gas production recovery patterns were then determined. The 50 day SRT inoculum source was maintained at 35°C, but was capable of metabolizing about 1000 mg l-m-day of acetate even when incubated at 25°C. Figure 12 shows the results. At 35°C, there was no significant reduction in gas production after addition of chloroform. However, at 25°C, gas production dropped to 50% of normal and required 15 days for complete recovery.
REFERENCES
APHA (1975) Standard Methods for the Examination o f Water and Wastewater, 14th edition. American Public Health Association, Washington, D.C. Baresi L., Mall R. A., Ward D. M. and Kaplan I. R. (1978) Methanogenesis from acetate: enrichment studies. Appl. envir. Microbiol. 36, 186. Bauchop T. (1967) Inhibition of rumen methanogenesis by methane analogues. J. Bact. 94, 1971. Bouwer E. J. and McCarty P. L. (1983) Transformations of 1- and 2-carbon halogenated aliphatic organic compounds under methanogenic conditions. Appl. envir. Microbiol. 45, 1286-1294. Miller T. L. and Wolin M. S. (1974) A serum bottle modification of the hungate technique for cultivating obligate anaerobes. Appl. Microbiol. 27, 985. Owen W. F., Stuckey D. C., Healey J. B. Jr, Young L. Y. and MeCarty P. L. (1979) Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Wat. Res. 13, 485. Stickley D. P. (1970) The effect of chloroform in sewage on the production of gas from laboratory digesters. Wat. Pollut. Control 69, 585. Swanwick J. D. and Foulkes M. (1971) Inhibition of anaerobic digestion of sewage sludge by chlorinated hydrocarbons. Wat. Pollut. Control 70, 58. Thiel P. G. (1969) The effect of methane analogues on methanogenesis in anaerobic digestion. Wat. Res. 3, 215. Yang J. and Speece R. E. (1986) The effect of engineering control on methane fermentation toxicity response. J. Wat. Pollut. Control Fed. In press. Yang J., Speece R. E., Parkin G. F., Gossett J. M. and Kocher W. M. (1980) The response of methane fermentation to cyanide and chloroform. Prog. Wat. TechnoL 12, 977.
APPENDIX
CONCLUSIONS On the basis of the results of these studies reported herein, the following conclusions are shown: (1) An acetate enriched, methanogenic culture was able to acclimate to the presence of various concentrations of chloroform while fermenting acetate to methane. (2) The severity and duration of inhibition of methane fermentation by a slug dose of chloroform was proportional to the toxicant concentration. (3) The system with the longest SRT could recover from the highest slug dose of chloroform. (4) Recovery from inhibition was directly related to biomass concentration. (5) Methane fermentation inhibition was directly related to the exposure period of biomass to chloroform and toxicity reversal was demonstrated
The following equation was used for the partitioning coefficient:
c, M,V,= Cs
Mr,
M,
II,
X V , - M , Vt
where: K = partition coefficient Ct = chloroform concentration in liquid phase after equilibrium (rag 1- l ) Cs = chloroform concentration in gas phase after equilibrium (mg 1- i) Mt = mass of chloroform in liquid phase after equilibrium (mg) Ms = mass of chloroform in gas phase after equilibrium (nag)
VI = volume of liquid phase (1.) V 8 = volume of gas phase (1.) X = concentration of chloroform initially added to liquid phase (rag 1- l ).
0043-1354/86$3.00+0.00 Copyright © 1986PergamonJournals Ltd
THE EFFECTS OF CHLOROFORM TOXICITY ON METHANE FERMENTATION JOSEPH YANG and R. E. SPEECE Environmental Studies Institute, Drexel University, Philadelphia, PA 19104, U.S.A. (Received October 1985) Abstract--Tbe methanogenic phase of anaerobic digestion is often considered to be the most sensitivestep in the overall process. Chloroform, a model industrial toxicant was assayed with methane fermentation, acetate enrichment cultures. Commonly, microbial toxicity response studies have observed the toxicity response over a relatively short time span, on the order of a few hours or days. In addition, cessation of observed cell function has often been assumed to be equated with death of the cells. The purpose of this study was to determine the toxicity response under various conditions over prolonged periods, on the order of months, with major emphasis on the recovery pattern. The following parameters were observed: concentration of toxicant, solids retention time, biomass concentration, toxicant exposure time, cell age, toxicant administration pattern and temperature. The methane fermentation culture was able to acclimate to the presence of chloroform while fermenting acetate to methane. Inhibition of unacclimated cultures was noted at 0.5 mg I-l, but with acclimation 15 mg I-l could be tolerated. There was a clear correlation between dose and inhibition. Recovery from inhibitionwas a function of SRT and biomass concentration. Younger cells were more resistant than older cells. The duration of chloroform exposure was directly related to the degree of residual inhibition. Reduced temperature delayed recovery of gas production from slug doses of chloroform. Radio tagged chloroform studies indicated that recovery of methane production occurred due to acclimation of the methanogens to the presence of chloroform and not from the disappearance of chloroform by microbial degradation. Key words--chloroform, toxicity, methane, fermentation, anaerobic digestion
INTRODUCTION The anaerobic digestion process for waste treatment is a mixed culture system which converts organic wastes to methane gas under anaerobic conditions. Normally, methane fermentation occurs satisfactorily in anaerobic sludge digestion and has an excellent potential for the treatment of warm industrial wastewaters. However, a number of features in regard to its response to toxicity have to be identified in order to insure reliable process performance. Commonly, microbial toxicity response studies have observed the toxicity response over a relatively short time span, on the order of a few hours or days. In addition, cessation of the observed cell function has often been assumed to be equated to death of the cells. The purpose of this study was to determine the response of the acetate conversion to methane step of anaerobic digestion under various toxicity conditions over prolonged periods, on the order of months, with major emphasis on the recovery pattern. The parameters which affect the toxicity of foreign substances are not only related to the kind of toxicant substances, but are also related to other factors. Some factors which can affect toxicity response are: concentration of toxicant, solids retention time (SRT), biomass concentration, toxicant exposure time, cell age, toxicant administration pattern, and temperature. These factors were assayed in this study.
The methanogenic phase is often considered to be the most sensitive step in the anaerobic digestion process. Therefore, it was determined to explore the response of acetate utilizing methanogens to a sample toxicant, chloroform, which is widely used industrially and is found in industrial waste effluents. Chloroform is very toxic to methane fermentation. Stickley (1970) reported 0.01 mg 1-I or more of chloroform in the sewage is likely to have an adverse effect on sludge digestion. Swanwick and Foulkes (1971) found chloroform to be most toxic to the anaerobic digestion of sewage sludge when compared with five other chlorinated hydrocarbon solvents including carbon tetrachloride and l,l,l-trichloroethane. Bauchop (1967) and Thiel (1969) used chloroform as a special inhibitor in studying the inhibition of methanogenesis owing to its being an analogue of methane. Swanwick and Foulkes (1971) concentrated on studying chloroform in anaerobic digestion due to its toxicity and the frequency with which it had caused difficulties in anaerobic digestors in Britain.
PROGRAM OF STUDY Seven sets of experiments were designed to evaluate the response of acetate methane fermentation to chloroform toxicity. These experiments were designed primarily to elucidate the role which the
1273
1274
JOSEPH YANG and R. E. SPEECE
following process design and control parameters play in toxicity response: (!) (2) (3) (4) (5) (6) (7)
Toxicant concentration SRT Biomass concentration Toxicant exposure time Cell age Toxicant administration pattern Temperature.
In this section, only a general outline of the program of study for each experiment is given. The re!eyant procedures for each experimental study are presented in each respective subsection of the results section to minimize redundancy.
Toxicity response according to toxicant concentration The response of methane production in semicontinuously fed methanogenic cultures to one-time, slug doses of chloroform was determined. Chloroform concentration was the variable parameter. With 50 day S R T systems, the microorganisms were exposed to 0.5, l, 2, and 2.5 mg l -t of chloroform. With 25 day S R T systems 0.5, l, and 2.5 m g l -l of chloroform concentration was assayed. With 12.5 day S R T systems, 0.25, 0.5, and 2.5 m g l -~ were assayed.
Toxicity response according to S R T In these studies, the experiment was similar to Item 1 above. However, S R T was the variable parameter. Chloroform concentrations tested at three S R T levels were 1 and 2 . 5 m g l -l.
Toxicity response according to biomass concentration Three different biomass concentrations were prepared in serum bottles and exposed to a range of concentrations of toxicant. One set had a biornass concentration in the serum bottles which was the same biomass concentration (925 mg l -t) as in the 50 day S R T inoculum culture. A second set had double this concentration, while the third set contained half of the biomass concentration of the 50 day S R T inoculum culture.
Toxicity response according to toxicant exposure time The cultures were exposed to a given chloroform concentration for the desired exposure time. The cells were then centrifuged, and the supernatant was withdrawn and replaced by unadulterated supernatant. Subsequently the gas production response was noted.
Toxicity response according to cell age The age of cells is reflected in the SRT. However, S R T reflects not only age of cell, but also biomass concentration. With a different SRT, both age and biomass concentration in reactors are different when loading rate was kept constant at 1000 mg l-l-day of acetate. Therefore, in these toxicity studies the effects of cell age on toxicity response were separated from the effects of biomass concentration. When assaying
the effect of cell age on toxicity, the reactors were adjusted to contain equal biomass concentrations with only cell age as a variable. They were operated as batch reactors subsequently.
Toxicity response according to toxicant administration patterns The chloroform was administered in two feeding patterns. Continuous dose of chloroform in the feed resulting in a gradual build-up in the culture. Slug dose to the culture resulting in first order washout.
Toxicity response according to temperature Two incubation temperatures were assayed, 25 and 35°C, at 50 day SRT. A slug dose of 1 m g l -~ of chloroform was added to both. MATERIALS AND METHODS
Inoculum source and feeding solution Methanogenic cultures enriched on acetate as the sole organic-carbon source (exclusive of 10mgl -~ of cysteine) were used in this study. A 4001. stainless steel CSTR inoculum digester with semi-continuous feeding and wasting, was maintained at a 50 day hydraulic and solids retention time and fed 1 g l-l-day of acetic acid. Glass carboys were used to culture the 25 and 15 day SRT inocula. All three inocula sources were operated in excess of three SRT before being used in this study. A defined nutrient salt solution, described elsewhere was used to maintain the inocula (Yang and Speece, 1986). Serum bottle technique The serum bottle technique as developed by Miller and Wolin (1974) and modified by Owen et al. (1979) was further modified for this study. The 50ml culture volume was anaerobically transferred into oxygen-free bottles of 125 ml volume. Once seeded, the serum bottles were operated in either batch or semi-continuous mode. After preliminary replicate experiments established the close replicability of results (the range of gas production variation for six replicates averaged < 10% with a maximum of 20%) all assays were run without further replication. The serum bottles were shaken once daily and incubated at 35°C except for the one assay indicated at 25°C. In a semi-continuous study, the serum bottles were maintained at a given SRT, the same SRT as the inoculum digesters, by daily withdrawal of the requisite volume of mixed contents and replacement with the nutrient salt solution using hypodermic syringes. Gas production was measured daily. The acetic acid concentration was stoichiometrically restored daily to 1000 mg 1-~ by adding glacial acetic acid with a microliter syringe in proportion to the volume of methane produced the previous day. In a batch study, no wasting of mixed contents or feeding of nutrient salt solution was conducted. Only gas production was measured and the acetic acid concentration was stoichiometrically restored to 1000 mg 1-I each day based on the volume of methane produced. ANALYTICAL PROCEDURES
Gas measurement Gas production in the serum bottles was measured by displacement of an acidified salt solution in a 100 ml graduated cylinder connected to a hypodermic needle which pierced the serum cap. Volatile suspended solids The biological solids were measured as volatile suspended solids (VSS). The analysis was performed according to
Effect of chloroform toxicity on methane fermentation Standard Methods (APHA, 1975). Glass fiber filters were put in a 550°C oven for 15 ruin before filtration to insure no volatile matter was present in the filters. Chloroform measurement The chloroform concentration in the cultures was measured by gas chromatography. A Shimadzu C_~-6 AM gas chromatograph with a glass column 3 ft x 3 mm i.d. containing packing material OV- 101 on 80/100 Supelcoport supplied by Supelco Inc., was used. Helium was employed as the carrier gas. The flow of carrier gas was 40 ml rain- 1. The injection temperature was 150°C and the column temperature was 85°C. Radioactive tagged chloroform analysis The radioactively tagged chloroform, [ ~4q was purchased from New England Nuclear, Boston, Mass. One ml of radioactively tagged chloroform sample was diluted into 10ml of liquid scintillation cocktail in plastic vials, then vigorously shaken. Counting was done with a Beckman LS-100C Liquid Scintillation System. The time for counting was set at 50 rain. The efficiency error was set at 1%. The samples were tagged at the level of 1000cpm. The background was 60cpm. A 48% counting efficiency was documented. RESULTS AND DISCUSSION Toxicant concentration Three SRT (50, 25, and 12.5 days) ranges were maintained in the serum bottles with respective VSS levels of about 925, 780, and 515 mg 1-l. A specified volume of mixed contents was wasted (1, 2, and 4 ml per day) and the same volume of nutrient solution was fed each day to maintain each given SRT. Gas production was measured daily. Several days of gas production were recorded prior to injection of the indicated slug doses of chloroform to establish a base line. The partitioning of chloroform between the liquid and gas phases was experimentally determined to be 3.1 at 35°C with 50 ml of liquid volume in a 125 ml serum bottle. The theoretically calculated partition coefficient was 2.9 under these conditions. The equations used for this calculation are shown in the Appendix. For our conditions, 68% of the initial injection of chloroform remained in the liquid phase after equilibration.
1275
The tendency of chloroform to adsorb/absorb to the biomass was evaluated by injecting radio-tagged chloroform into cultures containing 0 (initially centrifuged supernatant), 1, 2, and 4 times the biomass concentration of the 50 day SRT inocula (925 mg 1- ]) and allowing 6 days of equilibration. Then the cultures were centrifuged and the radioactivity in the supernatant determined. The control (initially centrifuged supernatant) was 4300 cpm. The 1 x concentration of biomass was 3800 cpm (88%), while the 2 x and 4 x concentrations of biomass were 3500 cpm (81%). In view of the fact that the counting precision was 200 cpm, these results indicate that adsorption/absorption of chloroform by the biomass reduced the liquid phase chloroform by 12% in 50 day SRT cultures. Figure 1 shows the results of chloroform toxicity on 50 days SRT methanogens. The chloroform concentrations shown in this study are the concentration initially injected into each system. Chloroform volatility caused it to be stripped by the gas production. The concentration of chloroform therefore decreased with time by gas stripping and liquid washout. Figure 2 shows how the calculated chloroform concentration would change by stripping due to gas production and liquid washout after 2 mg 1- l chloroform was injected into a 50 day SRT serum bottle. This calculated curve was verified by radioactively tagged chloroform experiments. A small difference in chloroform concentration can have a disproportionately large effect on recovery of methane production if the gas stripping ceases due to cessation of methane production as shown in Fig. 1 comparing a chloroform dose of 2 and 2.5 mg 1- i. A prolonged period of zero gas production would tend to lead one to the conclusion that the methanogens were "dead". However, the rapid gas production recovery from 2.5mgl -j would suggest the system biomass remained viable during this pro-
~, 2 m g / t chloroform injected into the 50 day SRT Serum Bottle -.. 2 0
Chloroform ~. 40
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=E o 1.5
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Fig. 1. Gas production response to chloroform slugs---50 day SRT,
0
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Fig. 2. Calculated chloroform concn in liquid phase 2mgl -l.
1276
Joseph YANO and R. E. SPF.ECE 15mg/t Chloroform •
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--30
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Fig. 3. Radioactive chloroform and gas production vs fime--1.5 m g 1-t.
Fig. 5. Effect of biomass conch on response to c h l o r o f o r m - 2mgl-L
longed period of zero gas production with metabolism blocked until detoxification and/or acclimation to the chloroform occurred. With the hypothesis that negligible bidtransformation was occurring, the chloroform concentration in a 50 day SRT digester was calculated for a 20 day period based upon the actual daily gas production and liquid wastage after a slug dose of 1.5 mg 1-l. During this same period carbon 14 tagged chloroform was spiked into the reactor and the radioactivity in the effluent samples was counted daily. As shown in Fig. 3, there is close agreement between the actual chloroform concentration based upon radioactivity in the effluent and that calculated on the assumption that biotransformation is insignificant and only gas stripping and liquid washout account for chloroform loss. Bouwer and McCarty (1983) demonstrated anaerobic metabolism of chloroform at 0.1 mg 1- ~ concentrations, which is 1/15 of the concentrations used in this study. Radio tagged chloroform studies with concentrated biomass, referred to previously showed the same supernatant radioactivity after 6 d~ys with 2 x and 4 x biomass Concentrations. If bidtransformation of chloroform was occurring, the radioactivity in the supernatant of the 4 x biomass concentration should have been less than in the 2 x .
12.5 day SRT in Fig. 4. Figure 4 shows that the 50 day SRT gas production recovered to normal faster than the 25 or 12.5 day SRT system. Also, the methanogens at a 50 day SRT could recover from 2 . 5 m g l -~ of chloroform from which the methanogens at 12.5 and 25 day SRT did not recover. Generally, it is considered that the greatest SRT provides the greatest process stability in the presence of toxic shocks to the biomass. The chloroform toxicity responses for 50, 25, and 12.5 day SRT semi-continuous systems are summarized in Table 1. In this case, the advantage of extended SRT in minimizing chloroform inhibition was demonstrated by increased gas production recovery rates at 50 days when compared with 25 and 12.5 day SRT, except for the case of 0.5 mg 1- ~ where all recovery times were equal. There are three factors which express the differences among SRT. One factor is biomass. In this study, the biomass at 50, 25, and 12.5 day SRT digesters was 925, 780 and 515mgl -~ respectively. The second factor is age of cell. The age of organisms in a digester is reflected in SRT. The third factor is the washout of toxicant and biomass. The concentration of toxicant is washed out faster in low SRT system. Therefore, this would tend to reduce inhibition.
SRT
Biomass concentration
In this study, gas production cross plots for 1 mg 1- l of chloroform are plotted for 50, 25, and 1 mglI Chloroform
~,
-~ 40
"h
To achieve the desired biomass concentration, 100ml of methanogenic culture was anaerobically transferred from the 50 day SRT inoculum culture into serum bottles. These bottles were then centri-
a
Table 1. Recovery time for chloroform toxicity in batch and sexni-eontinuous systems
"D-" =° o_
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Fig. 4. SRT response to chloroform slug--1 m g I-L
Chloroform cohen (mgl -~ )
Batch
0.25 0.5 1.0 2.0 2.5
--2.5 4 5
50 -3 4 7 60
25 -3 11 -did not produce gas for 60 days
12.5 0 3 25 -did not produce gas for 37 days
Effect of chloroform toxicity on methane fermentation fuged at 4000 rpm for 15 min and 50 ml of cell free supernatant fluid was withdrawn. Thus, these serum bottles then had double the biomass concentration of the standard 50 day SRT inoculum culture. When the serum bottles were seeded with 25 ml of methanogenic culture from the 50 day SRT inoculum culture and injected with another 25 mi of cell-free supernatant fluid, these serum bottles were considered to be at half the biomass concentration of the standard 50 day SRT inoculum culture. After preparing the serum bottles containing the desired biomass concentrations, 1000 mg 1-~ acetic acid was injected into each bottle. Then gas production was measured daily. The acetic acid level was stoichiometrically restored to 1000 mg I-l daily. Subsequently, various concentrations of chloroform were injected into each of the systems containing different biomass concentrations. During this experiment, no withdrawal of mixed contents or replenishment of nutrient solution was practiced in order to attempt to maintain the different relative biomass concentrations in the serum bottles. Three different biomass concentrations were evaluated in this phase. The results are shown in Fig. 5. When 2 mg I-l of chloroform was administered to the systems, the gas production in the system containing the highest biomass concentration recovered sooner than that of the lower biomass concentration systems. Toxicant exposure time
Serum bottles were seeded from the 50 day SRT inoculum culture and were injected with various concentrations of chloroform. After the desired exposure time (1 h, 1 day, etc.), the serum bottles were placed directly into a centrifuge and centrifuged at 4000rpm for 15min. They were then carefully inverted and the supernatant liquid phase was completely removed using a syringe. (In this study, toxicant exposure time refers to liquid phase only and ignores the 12% of toxicant absorbed on the biomass.) A 67/33% mixture of N2/CO~ was introduced while the supernatant liquid was withdrawn. Then, unadulterated, centrifuged supernatant fluid from the inoculum culture was injected into the serum bottle to replace the unadulterated supernatant fluid withdrawn. Daily gas measurement and stoichiometric acetate compensation were then continued as described before. During the experimental period, no mixed culture contents were wasted and no replenishment of nutrient solution was practiced. This phase of study was designed to elucidate the effect of toxicant exposure time on gas production recovery. Figure 6 shows the 1 h and 24 h exposure time responses to 50 and 200 mg 1-] of chloroform. When methanogens were exposed to 50mg 1-1 of chloroform the recovery of methane fermentation after 1 h chloroform exposure was faster than that for 24 h chloroform exposure.
1277
Chloroform 1 hr or 24 hrs Exposure
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Fig. 6. Effect of exposure time on response to chloroform. When the chloroform was removed from the liquid phase by replacing with unadulterated supernatant fluid, the gas production recovered after the lag periods shown. This indicates chloroform was bacteriostatic, but not a bactericidal and bacteriolytic agent in the concentration ranges and exposure times assayed. These results indicate the potential importance of rapidly separating the toxicant contained in the liquid phase from the biomass which is one advantage of the "plug-flow" anaerobic filter. Yang et al. 0980) found that continuous feeding of unadulterated nutrient salt solution into anaerobic filters subsequently after a one-day slug dose of 300 mg l - mof chloroform allowed the gas production to recover within l0 days. Although the dilution capability of a CSTR (completely stirred tank reactor) is considered to be a major advantage, it is now clear that, in some cases, not toxicant dilution, but either rapid removal of toxicity from contact with the biomass or toxicant removal, ensures more rapid recovery than does initial dilution followed by slow washout of the toxicant from a CSTR. Effect o f previous exposure on response
Figure 7 shows the relative response of the methanogens after previous exposure to various chloroform concentrations. Separate serum bottles were spiked with 0.5, 1.5, and 2 . 0 m g l -m of chloroform. These systems were then operated at 50 day SRT with 2.5 m g l l Chloroform
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Fig. 7. Effect of previous exposure to chloroform on reinjection response.
1278
JOSEPh YAr~o and R. E. SPEECE
daily restoration of the volatile acids concentration to 1000mg1-1 by stoichiometric supplementation of acetic acid in proportion to the previous days' methane production. After all of the systems had recovered to normal gas production, 2.5 mg1-1 of chloroform was reinjected into all cultures, including a control which had not previously been exposed to chloroform. As shown in Fig. 7, the system with the most protracted recovery time was the unacclimated control. The culture previously acclimated to 0.5 mg 1-1 chloroform had the second most protracted recovery. The culture previously exposed to 1.5 mg 1-~ chloroform demonstrated the shortest recovery period. This indicates that previous exposure of the methanogens to an "optimum" chloroform concentration facilitates acclimation which is manifested in reduced inhibition from subsequent exposures to chloroform.
Ce//age Cell-free supernatant fluid was used to dilute the biomass concentration in the 50 day SRT serum bottles down to the same VSS concentration as that found in the 12.5 day SRT serum bottles. The VSS concentration in the 50 day SRT inoculum culture was about 925 mg 1-1 and this was diluted down to 515 mg 1-l which was the biomass concentration in the 12.5 day SRT systems. Then the toxicity response pattern was evaluated in batch systems with no wasting of the mixed contents but stoichiometric restoration of acetate daily to 1000 mg 1-1. The results are shown in Fig. 8. With 1.5mg1-1 chloroform injected into the methanogenic cultures, the gas production in the 12.5 day $RT system took about 9 days to fully recover to the background rate, but the same biomass concentration of 50 day SRT methanogens had only recovered to 50% of the background rate after 17 days. Therefore, the younger cells of 12.5 day SRT were more robust and less adversely affected by chloroform than were equal biomass concentrations of 50 day SRT cells. Baresi et aL (1978) found H: and formate inhibition of methane fermentation from acetate were also related to the age of the enrichment culture.
IChloroform io
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70
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Fig. 9. Effect of gradual build-up of chloroform on response---50 day SRT. Toxicant administration pattern
In this phase of the investigation the chloroform toxicity response was compared for slug doses vs gradual administration in the feed. The gradual administration of toxicant simulates a waste stream of constant toxicant concentration being fed to a CSTR. With gradual administration of chloroform concentrations, the 50 day SRT culture could tolerate about 15 mg !-1 chloroform in the feed as shown in Fig. 9. However, with the 25 day SRT culture, only 5 mg !- l of chloroform could be tolerated in the feed as shown in Fig. 10. With the 12.5 day SRT culture, Ci°r°f°rm l e° c~o
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'
55
11. Effect o f g r a d u a l b u i l d - u p o f c h l o r o f o r m r e s p o n s e - - 1 2 . 5 d a y SRT.
J
40
on
Effect of chloroform toxicity on methane fermentation ~ I mg/1. Chloroform
5O 4O
•
when the biomass was separated from the supernatant fluid containing chloroform. (6) Previous exposure to chloroform reduced inhibition due to subsequent reinjection of chloroform. (7) At equal biomass concentrations, younger cultures proved more robust and resistant to chloroform than older cultures. (8) Reduced temperature (25°C) significantly delayed recovery of gas production from slug doses of chloroform when compared to the normal incubation temperature (35°C).
__'o• • /..~---~-~~ /
z
3O
I
/
o cJ
2O
o
•
10 0
35 *C
~ - - - 25 *C 0
i
5
I i i 20 lo 15 TIME (days}
1279
I
I
25
30
Fig. 12. Effect of incubation temperature on toxicity response to chloroform--1 mgl -t. even 1 mg 1-1 of chloroform in the feed caused eventual failure of gas production as shown in Fig. 11. By comparison, a slug dose of only 2.5mgl -~ was devastating to all systems. Temperature
Toxicity response characteristics were investigated at two different incubation temperatures, i.e. 25 and 35°C. Serum bottles were maintained at 50 day HRT and SRT by daily wasting of 1 ml of mixed contents and replacement with 1 ml of nutrient solution. Acetate was stoichiometrically compensated daily to 1000mgl -l according to methane production. The systems were operated for 3 days prior to injection of chloroform in order to establish the background methane production rate. At this point the same concentration of chloroform was injected into bottles which were incubated at 25 and 35°C. The gas production recovery patterns were then determined. The 50 day SRT inoculum source was maintained at 35°C, but was capable of metabolizing about 1000 mg l-m-day of acetate even when incubated at 25°C. Figure 12 shows the results. At 35°C, there was no significant reduction in gas production after addition of chloroform. However, at 25°C, gas production dropped to 50% of normal and required 15 days for complete recovery.
REFERENCES
APHA (1975) Standard Methods for the Examination o f Water and Wastewater, 14th edition. American Public Health Association, Washington, D.C. Baresi L., Mall R. A., Ward D. M. and Kaplan I. R. (1978) Methanogenesis from acetate: enrichment studies. Appl. envir. Microbiol. 36, 186. Bauchop T. (1967) Inhibition of rumen methanogenesis by methane analogues. J. Bact. 94, 1971. Bouwer E. J. and McCarty P. L. (1983) Transformations of 1- and 2-carbon halogenated aliphatic organic compounds under methanogenic conditions. Appl. envir. Microbiol. 45, 1286-1294. Miller T. L. and Wolin M. S. (1974) A serum bottle modification of the hungate technique for cultivating obligate anaerobes. Appl. Microbiol. 27, 985. Owen W. F., Stuckey D. C., Healey J. B. Jr, Young L. Y. and MeCarty P. L. (1979) Bioassay for monitoring biochemical methane potential and anaerobic toxicity. Wat. Res. 13, 485. Stickley D. P. (1970) The effect of chloroform in sewage on the production of gas from laboratory digesters. Wat. Pollut. Control 69, 585. Swanwick J. D. and Foulkes M. (1971) Inhibition of anaerobic digestion of sewage sludge by chlorinated hydrocarbons. Wat. Pollut. Control 70, 58. Thiel P. G. (1969) The effect of methane analogues on methanogenesis in anaerobic digestion. Wat. Res. 3, 215. Yang J. and Speece R. E. (1986) The effect of engineering control on methane fermentation toxicity response. J. Wat. Pollut. Control Fed. In press. Yang J., Speece R. E., Parkin G. F., Gossett J. M. and Kocher W. M. (1980) The response of methane fermentation to cyanide and chloroform. Prog. Wat. TechnoL 12, 977.
APPENDIX
CONCLUSIONS On the basis of the results of these studies reported herein, the following conclusions are shown: (1) An acetate enriched, methanogenic culture was able to acclimate to the presence of various concentrations of chloroform while fermenting acetate to methane. (2) The severity and duration of inhibition of methane fermentation by a slug dose of chloroform was proportional to the toxicant concentration. (3) The system with the longest SRT could recover from the highest slug dose of chloroform. (4) Recovery from inhibition was directly related to biomass concentration. (5) Methane fermentation inhibition was directly related to the exposure period of biomass to chloroform and toxicity reversal was demonstrated
The following equation was used for the partitioning coefficient:
c, M,V,= Cs
Mr,
M,
II,
X V , - M , Vt
where: K = partition coefficient Ct = chloroform concentration in liquid phase after equilibrium (rag 1- l ) Cs = chloroform concentration in gas phase after equilibrium (mg 1- i) Mt = mass of chloroform in liquid phase after equilibrium (mg) Ms = mass of chloroform in gas phase after equilibrium (nag)
VI = volume of liquid phase (1.) V 8 = volume of gas phase (1.) X = concentration of chloroform initially added to liquid phase (rag 1- l ).