enzyme activity for monitoring the stability in a thermophilic anaerobic digestion of wastewater...

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JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 71, No. 4, 264-269. 1991 Enzyme Activity for Monitoring the Stability in a Thermophilic Anaerobic Digestion of Wastewater Containing Methanol MAKOTO YAMAGUCHI,* JOHN HAKE, YUICHI TANIMOTO, TAKAAKI NARITOMI, KAZUO OKAMURA, AND KIYOSHI MINAMI Advanced Technology Research Department, Institute of Technology, Shimizu Corp., 4-17, Etchujima 3-chome, Koto-ku, Tokyo 135, Japan Received 10 September 1990/Accepted 8 January 1991 To maintain good conditions in a thermophilic methane bioreactor treating methanol as the main substrate, microbial enzyme activity in the reactor was investigated. In a preliminary study, seven enzymes were tested for their suitability as indicators using an acid-former, 22a originating from a digester with low efficiency, Methanosarcina sp. (CHTI-55) and Desulfotomaculum nigrificans (Delft 74). Among the tested strains, the ac- tivities of seven enzymes were the highest in 22a. Acidic phosphatase (ACP), glutamic-pyruvic transaminase (GPT) and a-amylase (AMY) were chosen as hopeful indicators for lab-scale tests and their activities were measured at the optimum temperature of 55°C. In the lab-scale test, reactor failure was induced by nitrogen deficiency or addition of dimethyl disulfide (DMDS) as an inhibitor. ACP, GPT and AMY outperformed the conventional parameters as indicators of any instability in the process. Recently, attempts to treat wastewater using new anaero- bic fermentation techniques have attracted great interest. Anaerobic treatment offers a number of attractive advan- tages in the treatment of strong organic wastes such as the ability to treat high organic loads, production of a small quantity of excess sludge and production of methane gas as a useful energy source. However, a primary disadvan- tage of anaerobic treatment is the difficulty in maintaining the stability of the process, since many different organisms are involved in the treatment process and each has its role in a highly synergistic environment. Therefore, the process is sensitive to environmental changes in the reactor and disturbances in the microbial population balance often re- sults in the failure of the treatment. For anaerobic digestion to be a viable treatment alter- native, it is necessary to have an efficient indicator of the stability of the process. Conventional process parameters such as concentrations of volatile suspended solids (VSS), mixed liquor suspended solid (MLSS) and volatile fatty acid (VFA), gas composition and production, removal of total organic carbon (TOC), biochemical oxygen demand (BOD) and chemical oxygen demand (COD) have been used. One of these, VSS, is not an accurate indicator of biomass activity because it also measures non-viable bio- mass and non-biological solids. The other parameters de- termine the end-products of metabolism and are indirect measures of microbial activity. Direct measurements of activity have also been devel- oped. Ashley and Hurst have used the activity of acidic and alkali phosphatases (ACP and ALP respectively) to predict anaerobic digester failure (1) and found that these enzyme activities were related to the population of acid- forming bacteria. Chung and Neethling have used adenosine triphosphate (ATP) and dehydrogenase activity (DHA) to monitor microbial activity in an anaerobic digester (2, 3). ATP is a measure of total bacterial activity in a digester and cannot distinguish between population groups. Conversely, DHA is substrate specific and has the ' Corresponding author. potential to measure the activity of a particular group of organisms. These studies concluded that enzyme activity measures outperformed the traditional parameters in predicting digester instability. Previous research has been concerned with the mes- ophilic digestion of domestic sludges (1-3). Our labora- tory is presently studying the high-rate anaerobic digestion of evaporator condensate (EC) waste generated by the kraft pulp process, which includes methanol as the main carbon source (4-6). This paper describes an investigation into the measurement of microbial enzyme activity in a thermophilic bioreactor treating synthetic waste similar to EC waste. First, a feasibility study was completed to iden- tify enzymes which held the greatest potential for charac- terizing process behavior. The indicators chosen would be used in a lab-scale experiment to determine whether enzyme activity such as ACP, GPT and AMY outperformed con- ventional parameters. MATERIALS AND METHODS Organisms Methanosarcina sp. CHTI-55 (DSM 2906) and Desulfotomaculum nigrificans Delft74 (DSM574) were obtained from the German Collection of Micro- organisms, G6ttingen, Germany (7). The optimum growth temperature of both strains was 55°C. After acclimati- zation of a mixed culture from our reactor in bad condi- tion, 22a was taken by the roll-tube method and had pre- dominantly acid-formers. Enzymes and chemicals Enzyme activities were meas- ured with modification of clinical diagnostic kits from Iatron Laboratories (Tokyo). Chemicals for media were from Wako Pure Chemical Industries (Osaka). All other chemicals were from Sigma (St. Louis, USA). Media The synthetic medium composition for lab- scale digester is as follows (in grams per liter tap water): methanol 10, (NH4)2SO 4 1.0, K2HPO 4 0.75, KH2PO 4 0.5, MgSO4.7H20 0.24, residue from alcohol distillate (4) 0.6 ml, mineral salt solution 1 ml. The mineral salt solu- tion contained (g//): FeCIj.6H20 9.5, MnC12.4H20 0.7, 264

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Page 1: Enzyme activity for monitoring the stability in a thermophilic anaerobic digestion of wastewater containing methanol

JOURNAL OF FERMENTATION AND BIOENGINEERING Vol . 71, No . 4, 2 6 4 - 2 6 9 . 1991

Enzyme Activity for Monitoring the Stability in a Thermophilic Anaerobic Digestion of Wastewater Containing Methanol

M A K O T O Y A M A G U C H I , * J O H N HAKE, YUICHI T A N I M O T O , T A K A A K I N A R I T O M I , K A Z U O O K A M U R A , AND KIYOSHI M I N A M I

Advanced Technology Research Department, Institute of Technology, Shimizu Corp., 4-17, Etchujima 3-chome, Koto-ku, Tokyo 135, Japan

Received 10 September 1990/Accepted 8 January 1991

To maintain good conditions in a thermophilic methane bioreactor treating methanol as the main substrate, microbial enzyme activity in the reactor was investigated. In a preliminary study, seven enzymes were tested for their suitability as indicators using an acid-former, 22a originating from a digester with low efficiency, Methanosarcina sp. (CHTI-55) and Desulfotomaculum nigrificans (Delft 74). Among the tested strains, the ac- tivities of seven enzymes were the highest in 22a. Acidic phosphatase (ACP), glutamic-pyruvic transaminase (GPT) and a-amylase (AMY) were chosen as hopeful indicators for lab-scale tests and their activities were measured at the optimum temperature of 55°C. In the lab-scale test, reactor failure was induced by nitrogen deficiency or addition of dimethyl disulfide (DMDS) as an inhibitor. ACP, GPT and AMY outperformed the conventional parameters as indicators of any instability in the process.

Recently, at tempts to treat wastewater using new anaero- bic fermentat ion techniques have at t racted great interest. Anaerobic t reatment offers a number of at tractive advan- tages in the t reatment of strong organic wastes such as the abil i ty to treat high organic loads, product ion of a small quant i ty of excess sludge and product ion of methane gas as a useful energy source. However, a pr imary disadvan- tage of anaerobic t reatment is the difficulty in maintaining the stabili ty of the process, since many different organisms are involved in the t reatment process and each has its role in a highly synergistic environment . Therefore, the process is sensitive to environmental changes in the reactor and disturbances in the microbial popula t ion balance often re- sults in the failure of the t reatment .

For anaerobic digestion to be a viable t reatment alter- native, it is necessary to have an efficient indicator of the stabili ty of the process. Convent ional process parameters such as concentrat ions of volatile suspended solids (VSS), mixed liquor suspended solid (MLSS) and volatile fatty acid (VFA), gas composi t ion and product ion, removal of total organic carbon (TOC), biochemical oxygen demand (BOD) and chemical oxygen demand (COD) have been used. One of these, VSS, is not an accurate indicator of biomass activity because it also measures non-viable bio- mass and non-biological solids. The other parameters de- termine the end-products of metabol ism and are indirect measures of microbial activity.

Direct measurements of activity have also been devel- oped. Ashley and Hurst have used the activity of acidic and alkali phosphatases (ACP and A L P respectively) to predict anaerobic digester failure (1) and found that these enzyme activities were related to the popula t ion of acid- forming bacteria. Chung and Neethling have used adenosine t r iphosphate (ATP) and dehydrogenase activity (DHA) to moni tor microbial activity in an anaerobic digester (2, 3). ATP is a measure of total bacterial activity in a digester and cannot distinguish between popula t ion groups. Conversely, DHA is substrate specific and has the

' C o r r e s p o n d i n g a u t h o r .

potential to measure the activity of a part icular group of organisms. These studies concluded that enzyme activity measures ou tper formed the t radi t ional parameters in predicting digester instability.

Previous research has been concerned with the mes- ophilic digestion of domestic sludges (1-3). Our labora- tory is presently studying the high-rate anaerobic digestion of evapora tor condensate (EC) waste generated by the kraf t pulp process, which includes methanol as the main carbon source (4-6). This paper describes an investigation into the measurement of microbial enzyme activity in a thermophil ic bioreactor treating synthetic waste similar to EC waste. First , a feasibility study was completed to iden- tify enzymes which held the greatest potential for charac- terizing process behavior. The indicators chosen would be used in a lab-scale experiment to determine whether enzyme activity such as ACP , GPT and AMY outperformed con- ventional parameters .

M A T E R I A L S A N D M E T H O D S

Organisms Methanosarcina sp. CHTI-55 (DSM 2906) and Desulfotomaculum nigrificans Delft74 (DSM574) were obtained from the German Collection of Micro- organisms, G6tt ingen, Germany (7). The op t imum growth temperature of both strains was 55°C. After acclimati- zation of a mixed culture from our reactor in bad condi- tion, 22a was taken by the roll- tube method and had pre- dominant ly acid-formers.

Enzymes and chemicals Enzyme activities were meas- ured with modification of clinical diagnostic kits from Iatron Laborator ies (Tokyo). Chemicals for media were from Wako Pure Chemical Industries (Osaka). All other chemicals were from Sigma (St. Louis, USA).

Media The synthetic medium composi t ion for lab- scale digester is as follows (in grams per liter tap water): methanol 10, (NH4)2SO 4 1.0, K2HPO 4 0.75, KH2PO 4 0.5, MgSO4.7H20 0.24, residue from alcohol distillate (4) 0.6 ml, mineral salt solution 1 ml. The mineral salt solu- tion contained (g//): FeCI j .6H20 9.5, MnC12.4H20 0.7,

264

Page 2: Enzyme activity for monitoring the stability in a thermophilic anaerobic digestion of wastewater containing methanol

VoL. 71, 1991 ENZYME ACTIVITY TO MONITOR ANAEROBIC DIGESTION 265

COC12-6H20 0.17, CaCla.2H:O 0.7, ZnC12 0.7, CuC12. 2H20 0.18, H3BO 3 0.07, NaMoO4.2H:O 0.17, NiC12.6H20 0.84. In the ammonia deficiency experiment, ammonium sulfate was replaced by sodium sulfate so that influent sulfate concentrations remained the same throughout the experiment. In the dimethyl disulfide (DMDS) addition ex- periment, DMDS was added to the above medium by dissolving DMDS in methanol before adding it to the me- dium. The medium containing DMDS was then supplied to the bioreactor with stirring.

Screening to measure enzyme activities in each strain was performed in culturing bottles with 100 ml of culture media. The culture medium composition for Methanosar- cina sp. with modification of DSM318 medium consisted of the following (in grams per liter deionized water): KH2PO 4 0.3, NaC1 0.6, MgC12-6H20 0.1, CaC12.2H20 0.08, NH4CI 1.0, KHCO3 2.0, yeast extract 0.5, peptone 0.5, methanol 5.0, resazurin 0.001, cystein-hydrochloride 0.3, Na2S-9H20 0.3, 10 ml of vitamin solution (DSM141) and 10 ml of trace element solution (DSM318) (7). The cul- ture medium composition for D. nigrificans consisted of the following (in gram per liter deionized water): KH2POa 0.5, Na2SO 4 1.5, MgSO 4. 7H20 1.5, sodium Dt-lactate 7.0, Fe(NH4)2(SO4)2.6H20 0.001, yeast extract 5 and peptone 5 (7). The culture medium composition for strain 22a con- sisted of the following (in gram per liter deionized water): K2HPO4 0.3, KH2PO4 0.2, NH4Cl 0.5, MgSO4-7H20 0.5, CaC12.2H20 0.3, NaCl 2.0, NaHCO3 1.0, FeSO4.7H20 0.002, Na2S. 9H20 0.3, cystein-hydrochloride 0.4, yeast ex- tract 2.0, peptone 2.0, resazurin 0.001, methanol 10, 10 ml of vitamin solution (DSMI40) (7), 10 ml of trace element solution (DSM140) (7). The anaerobic techniques for me- dium preparation and cultivation were those of Hungate with modification (8). The media were prepared under one atmosphere of pressure with a gas composition of 80%0 N:, and 20%0 CO2. The pH was adjusted to 7.0 and the growth temperature was 55°C.

Fermentation in lab-scale digester Thermophilic an-- aerobic digestion took place in a 200-• bioreactor (working volume: 140/) with a fixed bed constructed of pumice stones (4). The reactor was seeded with sludge from it methanogenic digester in methanol-rich medium (9). The pH of the effluent was monitored at the outlet of the digester and controlled at a desired pH with 0.5 N NaOH by using a pH controller. The effluent was recirculated to maintain pH and concentrations of methanol and nutrients in the reactor. The feed rate was set at 50 l/d and the recycle ratio was 1 : 1. In the DMDS addition experi- ment, we used a 1-! bioreactor (working volume: 700 ml) and the system was similar to that of a 200-• bioreactor. The feed rate was set at 300 ml/d.

Cell-free extract preparation Samples from reactor effluent and culture bottle were centrifuged (12,000 × g for 15 min). Supernatant was filtered with a 0.45/~m pore-sized Chromatodisc filter and used for VFA, methanol, am- monia, sulfate and TOC analyses. Exactly 100/11 of centri- fuged solids were mixed with 300 H1 of deionized water and sonicated for 5 min in an Omron biorupor (Tokyo) set at 130 W and 20 kHz. The sonicated sample was centrifuged with micro-test tube (2,000 x g for 10 min) and the superna- tant was retained for enzyme activity analysis.

Enzyme analysis In the feasibility portion of the ex- periment, 7 enzymes in the cell-free extract were tested: ACP, ALP, GPT, glutamic-oxaloacetic transaminase (GOT), ~-amylase (AMY), cholinesterase (ChE), and y- glutamyl transpeptidase (y-GTP). Three of these, ACP,

GPT and AMY were used in the lab-scale digester experi- ment. The kit procedures for ACP and ALP were col- orimetric analyses based on methods developed by Kind (10) and King (11), and the color reagent (CR) was 4- aminoantipyrine. Kit procedures for GPT and GOT were colorimetric with the analyses based on the methods of Reitman and Frankel (12), and the CR was 2,4-dinitro- phenylhydrazine. The AMY procedure was based on a method developed by Marshall (13) and the CR was N- ethyl-N-sulfopropyl-m-toluidine and 4-aminoantipyrine. The kit procedures for 7-GTP were colorimetric with the analysis based on the method of Shimamoto (14) and the CR were p-xylenol and sodium metaperiodate. The ChE was colorimetric with the analysis based on the method of Okabe (15) and the CR used was 4-aminoantipyrine. The absorbance of colorimetric indicators was measured with a Hitachi 220S spectrophotometer (Tokyo). A wavelength of 500 nm was used for ACP, ALP, GPT, GOT and ChE enzymes. The wavelength used for 7-GTP was 635 nm.

Analytical techniques The methane content of the gas was determined by a gas chromatography column (GC- 7A, Shimadzu Corp., Co., Kyoto), TOC was measured by a TOC analyzer (TOC-500, Shimadzu Corp.). Methanol and VFA such as acetic and propionic acids were analyzed by a high-pressure liquid chromatographic procedure using an ion-exchange column (KC-811 and KC-810p, Showa Denko K.K., Tokyo). Samples were analyzed by measuring their refractive indexes (RID-300, Japan Spec- troscopic Co. Ltd. (JASCO), Tokyo) and an Intelligent UV/VIS detector (870-UV, JASCO) and using a post-col- umn reaction with a pH indicator, bromothymol blue (BTB), for fatty acid analysis (9). An IC-500 ion chromato- graphic analyzer (Yokogawa Electroc Corp., Tokyo) with a conductivity detector was used for measuring sulfate and ammonium ion concentrations. Determination of MLSS in the effluent was carried out by filtering with a pre- weighed 0.5/~m glass fiber filter in the modified testing method of Japanese Industrial Standard (JIS K 0102).

RESULTS

Enzyme screening A feasibility study was performed to determine which enzymes held the greatest potential as biochemical indicators of digester stability. This was de- cided on the basis of activity difference among species and detectability. An environmental change in the reactor may shift the balance of populations or activities, among the bacterial groups in the digester. When such a shift occurs, changes in enzyme activity which reflect this disturbance would serve as a useful indicator of stability. For this reason, we focused on enzymes with large differences in ac- tivity among major groups of bacteria. Detectability was also a criterion; we selected enzymes whose activities were easily detected and measured.

Seven enzymes were tested using three species of bacte- ria (strains CHTI-55, Delft74 and 22a). These three species were chosen because they are representative of the major bacterial trophic groups found in our reactor. Methanogen- esis, VFA formation, and sulfate reduction are all proc- esses which occur in the digestion of EC wastes (4, 6), in- dicating the presence of methanogens, acid-formers and sulfate-reducers, respectively.

All three species were incubated in their respective cul- turing media for one week. The enzymes tested were ACP, ALP, GPT, GOT, AMY, ChE, and y-GTP at 35°C, 45°C, 55°C and 65°C, respectively. All enzyme activities were

Page 3: Enzyme activity for monitoring the stability in a thermophilic anaerobic digestion of wastewater containing methanol

266 Y A M A G U C H I ET AL. J. FERMENT. BIOENG.,

the highest at an incubation temperature of 55°C, about twice as efficient as that at 35°C and results at 65°C were similar to that at 55°C (data not shown). Feasibility study results at 55°C are displayed in Fig. 1. Strain 22a had significantly higher activities for all of the enzymes tested. Strain CHTI-55 had a slightly higher activity than Delft74 for GOT, but the former was much lower than the latter for other enzymes except ChE and AMY. Strain CHTI-55 and Delft74 had no ChE or AMY activity. ALP, ChE and y-GTP were detected in low levels, and they were dropped from further study. GOT was also removed as a candidate for further study because its biochemical function is simi- lar to that of GPT and their preliminary activity results were comparable. ACP, GPT and AMY were thus the chosen enzymes for the lab-scale portion of the experi- ment.

Change in digestion pattern caused by ammonia defi- ciency In the lab-scale experiment, conditions of low digester efficiency were induced by nutrient deficiency. The effects of ammonia deficiency are shown in Fig. 2. Influent ammonia concentration was initially reduced to one-tenth of the standard concentration on day 8 of the experiment and then to zero on day 24. Effluent levels of ammonia reached zero on day 16; this was the onset of nutrient deficiency. If we defined conditions of low digester effi- ciency as reduction in either gas production or MLSS, then a period of low efficiency began on day 26, when both parameters went into a steady decline. These parameters also closely corresponded to TOC removal (data not

shown). There was a ten day lag between nitrogen deficiency and the onset of low digester efficiency.

In the interim period, VFA concentrations and enzyme activities began to change. Acetic acid concentration began a modest increase at day 20 and remained at a level of about 15-20 mg/l until day 27, then there was a sharp in- crease. Propionic acid concentration began a steady rise shortly thereafter. Enzyme activities, however, proved to be more timely indicators. ACP and AMY activities in- creased slowly at day 15 and more sharply at day 18. ACP and AMY increased at the onset of nutrient deficiency, peaked at day 23-40 and 21 respectively and then gradually declined. Changes in GPT activity, however, began to go into a decline immediately after nitrogen deficiency and continued a steady decline. After the resupply of ammonia at day 46, the activity of ACP and AMY increased slightly, followed by a steady condition, while that of GPT re- covered rapidly.

The concentration of acetic and propionic acids followed

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FIG. 1. Enzyme feasibility study. The enzyme activities of three different strains of bacteria were measured at 55°C after culturing in s tandard media for one week. The enzyme were acidic phosphatase (ACP), alkali phosphatase (ALP), glutamic-pyruvic t ransaminase (GPT), glutamic-oxaloacetic t ransaminase (GOT), ~-amylase (AMY), cholinesterase (ChE) and y-glutamyl transpeptidase (GTP). The en- zyme activities were in Methanosarcina sp. strain CHTI-55 ( • ), D. nigrificans strain Delft74 (~) and isolated acid former strain 22a ( ) .

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FIG. 2. Effects of ammonia deficiency on operating parameters of an anaerobic digester, including changes in enzyme activity of the sludge. During the month before ammon ia reduction, a 200-I digester was operated continuously at a dilution rate of 0.36 d ~ and 55°C under s tandard conditions as stated in Materials and Methods. In- fluent ammonia was reduced to one-tenth of the standard concentra- tion on day 8 of this experiment and then was not supplied on day 24. From day 46, influent ammonia was resupplied. All samples for analyses were taken from effluent. Enzyme activities measured were acidic phosphatase (ACP), glutamic-pyruvic t ransaminase (GPT) and ~-amylase (AMY). Upper symbols: [3, ACP; e , GPT; = , AMY. Middle symbols: ~ , acetic acid; A, propionic acid; ±, methanol . Lower symbols: e , gas production; c~, ammonia ; ±, MLSS.

Page 4: Enzyme activity for monitoring the stability in a thermophilic anaerobic digestion of wastewater containing methanol

VOL. 71, 1991 ENZYME ACTIVITY TO MONITOR ANAEROBIC DIGESTION 267

TABLE 1. Effects of dimethyl disulfide (DMDS) on operating parameters of an anaerobic digester, including changes in

enzyme activity of the biomass

Time (d) 0 4 8 11 13

Gas production (l/d) 2 2 1.9 1.4 1.8

DMDS (g/l) a 0 0.1 0.3 0.3 0.3

Effluent MeOH (mg//) 0 0 0 920 43 A.A. (mg//) 0 0 100 1,890 1,890 P.A. (rag//) 0 0 0 0 70 ACP (O.D.) 0.118 0.183 0.278 0.312 0.317 GPT (O.D.) 0.561 0.504 0.409 0.384 0.351 AMY (O.D.) 0.127 0.354 1.077 0.858 0.800

Condition: A one-liter digester was operated continuously at a dilu-. tion rate of 0.43 d ~ and 55°C under standard conditions as stated in Materials and Methods, during one month before DMDS addition. DMDS of 0.1 g/l was added to influent on day 1 of this experimen! and DMDS concentration was increased from 0.1 g/I to 0.3 g/I on day 8.

DMDS influent concentration. A.A.: Acetic acid; P.A.: propionic acid; MeOH: methanol. Enzyme activities measured were acidic phosphatase (ACP),

glutamic-pyruvic transaminase (GPT) and ~-amylase (AMY).

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the same cycle as ACP and AMY, with a time lag of ap- proximately 10-15 d (Fig. 2). It indicated that the behavior of these enzymes closely corresponded to the formation of VFA. The behavior of GPT and MLSS in the effluent caused by nitrogen deficiency and resupply was almost the same (Fig. 2). It also indicated that the behavior of both in- dicators corresponded closely to each other.

Change in digestion pattern caused by inhibitor addi- tion (DMDS) Under a nitrogen deficient condition, low digester efficiency was detected in its early stages by measur- ing enzyme activity. However, it is possible that the varia- tion in enzyme activity was directly caused by a deficiency of nutrients such as ammonia . To better understand the relationship between low digester efficiency and enzyme ac-- tivity, low digester efficiency was induced by other factors.

The results from a bioreactor, treating evaporator con- densate waste generated by the kraft pulp process, indi-- cated that the accumulat ion of DMDS in the digester was primarily responsible for the low process efficiency (6). Results from the cont inuous operation of a 1-I bioreactor with DMDS addit ion to influent are shown in Table 1. Dur- ing the week after the DMDS concentrat ion of 0.1 g/l was added to influent on day 1 of the experiment, gas produc- tion was stable and, VFA and methanol were not leaked into the effluent. Digester efficiency was apparently stable. The enzyme activities of ACP, AMY and GPT, however changed slightly (Table 1, day 4). Shortly after the DMDS influent concentrat ion was increased from 0.1 g/l to 0.3 g/I on day 8 of the experiment, a slight decrease in gas produc- tion and the format ion of acetic acid were observed as well as a variation in these enzyme activities (Table 1, day 8). Next periods of clearly low digester efficiency characterized by a decrease in gas production, methanol leakage, and the formation of acetic and propionic acids, were observed (Table 1, days 11 and 13).

The behavior of these enzyme activities under these con- ditions of DMDS addit ion was similar to that in the nitro- gen-deficient condit ion. We could detect a variation in en- zyme activity earlier than other phenomena.

Reliability of enzyme-measuring system In order to maintain a high digester efficiency, it is important to detect

Time (d)

FIG. 3. Monitoring of enzyme actiwty in thermophilic meth- anogenesis. For cultural conditions, see legend to Fig. 2. lnfluent am- monia was reduced to one-tenth of the standard concentration on day 0 of this experiment and then was not supplied on day 11. From day 14, influent ammonia was resupplied. All samples for analyses were taken from effluent. Enzyme activities measured were acidic phosphatase (ACP) and glutamic-pyruvic transaminase (GPT). Upper symbols: e , gas production; [], acetic acid; <, ammonia. Lower symbols: G, ACP; o, GPT.

a bad digester condit ion at the earliest possible stage. By monitor ing these enzyme activities, we can succeed in de- tecting the prelude to trouble in the digester. As soon as a change of enzyme activity occurred under the condit ion of nitrogen deficiency, ammonium salt was resupplied to the bioreactor. This occurred on day 14 of the experiment (Fig. 3). Although acetic acid was detected in the effluent, the acetic acid was not produced early and the condit ion of the digester recovered before there was a decrease in gas production (Fig. 3). We observed similar phenomena in the digester fed with DMDS in the influent (data not shown).

DISCUSSION

Two divisions of bacteria may exist in our bioreactor: eubacteria and archaebacteria. Methanogens are archae- bacteria which have a metabolism that utilizes coen- zymes unique to methanogenesis, such as factor 420 and coenzyme M (16). However, these unique coenzymes and the other enzymes of methanogens are difficult to measure easily. These are other enzymes which are more easily meas- ured and are utilized by both groups or by only the eubacte- ria. AMY may be an enzyme utilized by only the eubacte- ria in the bioreactor. Methanogens can not utilize high- molecular-size carbohydrate. Because of this distinction, AMY has potential as an indicator of the activity of acid- forming bacteria. However, indicators of populat ion or ac- tivity shifts in digester ecology are not limited to enzymes peculiar to a single group of bacteria. Enzymes common to

Page 5: Enzyme activity for monitoring the stability in a thermophilic anaerobic digestion of wastewater containing methanol

268 YAMAGUCHI ET AL. J. FERMENT. BIOENG.,

both divisions, such as: phosphatases, ALP and ACP; transaminases, GPT and GOT; a peptidase, y-GTP; a dehydrogenase, ChE, also hold potential as indicators. One purpose of the feasibility study was to measure activity differential between groups. The greater the dif- ferential, the greater the sensitivity of enzyme activity as a measure of shifts in the balance of populations and activity. Differences in enzyme activity between groups may re- flect differences in metabolism.

ACP, AMY and GPT were desirable for further testing largely because their activities may be strongly associated with acid forming bacteria (Fig. 1). Periods of low efficiency in our reactor, defined by low gas production and reduced TOC removal, are characterized also by increased con- centrations of VFA. Similar conditions have been reported previously for both the digestion of domestic sludges (17, 18) and the treatment of evaporator condensate wastes (6). It was postulated that during such periods, the popula- tion and activity of acid-forming bacteria relative to methanogens increased. If this is the case, then we would expect ACP, AMY and GPT activities of the reactor bio- mass to increase during periods of low digester efficiency.

The behavior of ACP and AMY closely corresponded to the formation of VFA (Fig. 2). It is postulated that the acid-forming bacteria are more resistant to nitrogen deficient conditions and that the rise in ACP and AMY ac- tivities reflects a shift in the populat ion or activity balance from methanogens to acid-formers. The subsequent de- crease in ACP and AMY is probably due to an eventual decline in the populat ion or activity of acid-formers, when nitrogen deficiency becomes too severe. The VFA concen- tration followed this same cycle, with a time lag of approxi- mately 10-15 d (Fig. 2).

The consumption rate of sulfate also closely corre- sponded to low digester efficiency during the period of nitrogen deficiency (data not shown). Under these condi- tions effluent sulfate concentrat ion is an efficient indicator of instability. However, evaporator condensate waste ex- hibits a wide variation in sulfate concentration. Practically speaking, it would be difficult to monitor digester efficiency based on effluent sulfate concentrations or the enzyme activity of sulfate-reducing bacteria.

We expected GPT to behave similarly to ACP and AMY on the basis of the feasibility study results. Strain 22a showed much greater activity than the methanogens for these enzymes (Fig. 1). However, unlike ACP and AMY, GPT activity declined at the onset of nutrient deficiency. It recovered rapidly after the resupply of ammonia (Fig. 2). The reasons for this behavior are unclear. One possibility is that GPT is an amino transferase and bacteria may reduce production of this enzyme in low digester efficiency. This reduction might offset relative increases in the pop- ulation or activity of biomass. GOT also holds potential as a biochemical indicator. Feasibility study results for GOT and GPT were similar. Both enzymes are amino trans- ferases. These common characteristics lead us to believe that GOT would behave similarly to GPT and serve as a useful indicator of reactor instability.

Low digester efficiency caused by nitrogen deficiency was observed in experiments where DMDS was added to influent. These phenomena were also recognized in the overloading experiments (data not shown). These observa- tions suggest that variations in enzyme activities at the early stages of low digester efficiency are a common phe- nomena, and the mechanism of low efficiency in the digester caused by DMDS addition is similar to that for

nitrogen efficiency. Although ALP proved to be a successful indicator in pre-

vious investigations (1, 18), the ALP detection level was low and the changes in ALP activity were not significant unlike ACP, GPT and AMY in lab-scale experiments (Fig. 2, Table 1). Instability of ALP is probably not the cause because results of ALP tested at 35°C were similar to that at 55°C (data not shown).

In conclusion, the measurement of enzymes is well suited to the monitor ing of digester efficiency enabling the maintenance of opt imum conditions (Fig. 3). The micro- bial activity includes activities of several bacterial popula- tion groups involved in anaerobic sludge digestion. It may therefore be appropriate to perform several activity meas- urements simultaneously to assess the complex ecology of a digester. The measurement of ACP, GPT and AMY is the most direct way to detect a change in the biochemistry of the anaerobic digester to determine the stability of the process. In addition, consistent monitoring in the field seems necessary to determine the stability of microbial bio- mass and activity. Historical activity data is essential in determining the current status of an anaerobic sludge digester.

ACKNOWLEDGMENT

We thank Mr. Isao Fujimura and Mr. Tatsuya Sawai for their assistance in experiments.

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