Appl Microbiol Biotechnol (1987) 26:383--388 Applied Microbiology

Biotechnology © Springer-Verlag 1987

Hydrogen-dependent control of the continuous anaerobic digestion process

T. N. Whitmore, D. Lloyd, G. Jones, and T. N. Williams

Department of Microbiology, University College, Newport Road, Cardiff CF2 1TA, UK

Summary. Membrane inlet mass spectrometry was used to directly measure the concentrations of CH4 and H2 in a mesophilic (37 o C) completely mixed, laboratory scale, anaerobic digester, con- tinuously fed at a retention time of 7 days with a glucose (50 mM) mineral salts medium. When the digester was overloaded by an increase in the in- fluent substrate concentration, equivalent to 15.5 Kg (COD) m -3 (digester) day -1 the concentra- tions of H2 and short chain fatty acids increased with a concomitant decline in the pH: following an initial stimulation methanogenesis was inhi- bited. Regulation of the H2 signal from the mass spectrometer in a closed feedback loop by con- trolled addition of carbon source under a poten- tial overload condition, enabled the HE concentra- tion to be controlled around 1 ~tM and a high steady state rate of methanogenesis of 42 ~tM min-1 to be maintained; this is equivalent to 1.4 volumes of CH4 per culture volume per day. The hydrogen-dependent control system was also used to prevent inhibition of methanogenesis when the digester was subject to volumetric overloading potentially equivalent to a retention time of 1 day.

Introduction

The anaerobic digestion or biomethanation proc- ess by which waste organic matter is converted to biogas consisting of approximately 65% CH4 and 35% CO2 is, under normal operating conditions, self-regulating. Under overload conditions, how- ever, for example after a sudden increase in the influent substrate concentration (organic over-

Offprint requests to: D. Lloyd

load), or after an increase in the throughput of substrate (volumetric overload), the resulting acidification can cause process inhibition or even failure (Pohland and Bloodgood 1963; Archer 1983). For the process to become more widely ac- cepted, however, in preference to aerobic treat- ment systems, effective methods of process con- trol must be developed.

The importance of H2 as a central interme- diate in the process has been demonstrated re- cently. An elevated partial pressure of H2 in the biogas of stressed digesters was predicted from a mathematical model presented by Mosey (Mosey 1983; Mosey and Fernandes 1984). Archer et al. (1986) demonstrated increased H2 partial pres- sures in the biogas of a 6 m 3 digester treating bre- wery effluent subjected to shock loadings. Scott et al. (1983) and Whitmore et al. (1985) have shown rapid increases in the dissolved HE level of stressed laboratory scale digesters. Whitmore and Lloyd (1986a), have recently demonstrated the po- tential of using the dissolved H2 concentration, measured by using membrane inlet quadrupole mass spectrometry, as a method of process con- trol in laboratory scale fed batch thermophilic and mesophilic (Whitmore and Lloyd 1986b) anaerobic digesters. The present paper extends this work to a continuously fed laboratory diges- ter. It is demonstrated that the inhibition of me- thanogenesis, through overloading the digester, can be prevented by regulating the dissolved HE concentration by the controlled addition of sub- strate.

Materials and methods

Maintenance of anaerobic digester

Reproducibility between experimental runs was ensured by using a single laboratory scale anaerobic digester as a common

384 T . N . Whitmore et al.: Hydrogen control of anaerobic digestion

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source of culture samples for transference to the mass spec- trometer reaction vessel. The laboratory digester of working volume 5 1 was operated at a retention time of 10 days and maintained at 37 ° C. The original inoculum was a sample of pig slurry and the medium composition was as below:

Glucose, 9 .0g; MgSOa.7H20, 0.4; NH4C1, 1.4g; Na2HPO4, 3.0 g; KH2PO4, 1.5 g; trace mineral solution (Balch et al. 1979 with the addit ion of NiCI2, 0.4 g 1- ~) 10 ml and dis- tilled water 990 ml.

Mass spectrometry

A quadrupole mass spectrometer type SX200 and associated DPP16 digital peak programmer (VG Gas Analysis, Aston Way, Middlewich, Cheshire, UK) was used. The mass spec- trometer was linked to the 5 cm diameter stainless steel reac- tion vessel (40 ml total volume; 20 ml working volume open for gases) by a stainless steel probe (Bohfitka et al. 1983) (length 15 cm, i.d. 0.8 ram) fitted with a silicone rubber mem- brane (i.d. 0.8 ram; o.d. 1.7 mm) - - membrane covered 50 ~tm diam. inlet (Fig. la). The vessel was thermostated at 37°C and stirred at 300 r e v - m i n - ' . A N2 (80%) CO2 (20%) gas mixture (50 ml min-1) was passed over the surface of the culture in the vessel to maintain anaerobiosis. The gas was humidified by passage through a water jacket maintained at 37°C built into the system immediately adjacent to the reaction vessel cham- ber. The m/z ratios used to measure the concentrations of CH4 and H2 were 15 and 2, respectively; 6/2 values for equilibra- t ion in medium buffer were 3.3 min and 2.2 min, respectively. Samples (20 ml) were removed from the laboratory digester and transferred to the mass spectrometer reaction vessel, to which culture medium (composition as above) was supplied continuously at a rate of 0.12 ml h-~.

Gas product ion rates were determined from the following equation (Lloyd and Scott 1983; Lloyd et al. 1985):

L = K T L ,

where Vr is the production rate, K is the gaseous exchange constant, TL is the concentration of dissolved gas (K is l n 2 / tl/2, where tl/2 is the half-time for equilibration between gas and liquid in the absence of biological material).

Total gas production is proportional to the areas under the mass spectrometric traces.

Feedback control system

The analogue H2 signal from the SX200 was used to control a Gilson Minipuls 2 peristaltic pump which supplied substrate to the reaction vessel via a voltage level de tec tor /pump driver (Fig. lb) with the modification that TR1400 -0 .35 was re- placed by TIL206D). The same pump was used to remove an equivalent volume of culture from the reaction vessel.

Volatile fatty acid analysis

Acidified cell-free culture samples (5 ktl) were injected into a Pye 104 gas chromatograph equipped with a flame ionisation detector and column (100 cm.0.3 cm i.d.) packed with Chro- mosorb 101 (Jones Chromatography Ltd., Llanbradach, Mid Glamorgan, Wales, UK) maintained at 200 ° C.

Results and discussion

The effectiveness of the H2-dependent mass spec- trometric control system in the experiments de- scribed below was determined by allowing the anaerobic digester contents in the mass spectrom- etric reaction vessel to attain a steady state (i.e. a constant and reproducible rate of methanogene- sis), when supplied with 50 mM glucose mineral salts medium at a constant flow rate of 0.12 ml h -1 before perturbing the system.

Figure 2a shows the effects of increasing the loading rate to the digester contents by instanta- neously altering the influent glucose concentra- tion at zero time from 50 mM to 560 mM. The dis- solved CH4 concentration increased from 83 ~tM to 200 ~tM after 1 h and subsequently declined to 60 lxM towards the end of the experiment (20 h). Dissolved H2 increased from below the level of detectability (1 lxM) at approximately 15 min to a maximum of 38 pM around 14 h before decreas- ing. The pH of the digester contents fell from its initial value of 7.1 to 5.1 at 20 h.

Figure 2b shows the time course of the in- crease in volatile fatty acids during the experi- mental period. Acetic acid increased rapidly from its steady state level of 1.5 mM to 18 mM within 1 h and continued to increase at a lower rate until approximately 5 h; thereafter increasing to 49 mM at the end of the experimental period. The higher fatty acids showed a comparatively smaller proportional increase during the experiment from 0.3 mM to 15.1 mM (propionic) and 0.7 mM to 4.4 mM (butyric).

Comparison of Figs. 2a and 2b shows that the three phases in the acetic acid production are re- lated to the time course of changes in dissolved C H 4 . A decline in the initial rapid rate of metha- nogenesis occurred at around 1 h when acetic acid approached 2 0 m M ; this concentration would saturate the methanogenic reactions in ace- toclastic organisms as the Ks for acetate of this systems has been determined as 2.8 mM (Whit- more et al. 1985). Subsequently acetic acid and CH4 concentrations remained steady until a rise in the acetic acid concentration at around 5 h was paralleled by a decline in the CH4 concentration.

The initial response of methanogenic systems to surge load by the production of large quantities of acetic acid has been noted by Mosey (1982). The comparatively high concentrations of dis- solved H2 present later in the experiment accom- panying the decline in the methane concentration would tend to inhibit the thermodynamically un- favourable conditions for the oxidation of pro-

386 T .N . Whitmore et al.: Hydrogen control of anaerobic digestion

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Fig. 2. Effects of increased loading rate on an uncontrolled anaerobic digester. Influent glucose concentration was in- creased from 50 mM to 560 mM at time zero. (a) Hz and CH4 were continuously monitored in situ at m / z 2 and 15, respec- tively; pH was also measured directly. (b) Volatile fatty acids in samples withdrawn from the fermenter: (O) acetic acid, ( I ) propionic acid, ( A ) butyric acid. (c) Effects of increased loading rate on a controlled anaerobic digester. Influent glu- cose concentrat ion was increased from 50 mM to 560 mM at time zero. The dissolved hydrogen (not shown) never exceeded 1.3 IIM; the set point for the switch off of the feed supply pump was 1 I-tM H2. Accumulating fatty acids were assayed on samples removed from the fermenter: (O) acetic acid, ( I ) propionic acid, ( • ) butyric acid

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pionate and butyrate. The degradation of these fatty acids is believed to be accomplished by obli- gate proton-reducing acetogenic bacteria: Syntro- phomonas wolfei degrades butyrate (Mclnerney et al. 1981) and Syntrophobacter wolinii propionate (Boone and Bryant 1980). From the data of

Thauer et al. (1977) it can be calculated that the required H2 concentrations for the propionate and butyrate oxidations must be less than 0.1 I~M and 2.0p~M respectively for the free energy changes to become negative (with the concentra- tion of the acids 1 mM, hydrogen carbonate 25 mM, and temperature 37°C). The greater ac- cumulation of propionate (final concentration 15 mM) compared with butyrate (4.5 mM) in Fig. 2b can thus be explained by the energetically less favourable propionate oxidation reaction.

The effects of an identical overload to the di- gester contents to the one described above, but us- ing the H2-dependent control system to switch off the feed supply pump at a H2 threshold of 1 IIM is shown in Fig. 2c. The dissolved C H 4 increased from its steady state level of 82 ~M to 200 p.M within 2 h and remained at approximately this level throughout the subsequent course of the ex- periment. The dissolved H2 (not shown) fluc- tuated around the level of detectability of the in- strument (1 p,M) but never exceeded 1.3 I, tM. The pH of the digester contents fell slightly from the initial value of 7.2 (0.2 pH units in 20 h) but vola- tile fatty acids did not accumulate in contrast to those events observed during uncontrolled addi- tion of 560 mM glucose (Fig. 2b). In terms of the COD loading rate the continuous supply of glu- cose (Fig. 2a) is equivalent to 15.5 Kg (COD) m -3 (digester) day -1. Controlled COD loading rate was about 7.8 Kg (COD) m 3 day-1. From a recent literature survey Pfeiffer et al. (1986) disclosed the maximum loading rates of full scale conventional CSTR anaerobic digester to be around 10--11 Kg m -3 day-1. The successful H2-dependent control of the system described demonstrates the feasibil- ity of preventing digester failure through sudden increases in the organic loading rate.

In a previous paper (Whitmore and Lloyd 1986b) control of methanogenesis in a fed batch laboratory scale digester was only achieved when the H2 concentration was regulated at a threshold level of 0.25 ~tM by using a PTFE membrane inlet to the mass spectrometer. This paper has shown that control in a continuously fed system is more easily achievable because the accumulation of in- hibiting products (eg. fatty acids, H +) is dimin- ished.

The steady state rate of methanogenesis deter- mined from Fig. 2c of approximately 42 p,M min-1 is equivalent to a CH4 production rate (at STP) of 1.4m 3 m -3 day -1, compared with the maximum value of around 2 m 3 m -3 day -1 ob- tained from a worldwide survey of full scale con- ventional digesters (Melchior et al. 1982).

T. N. W h i t m o r e et al.: H y d r o g e n control o f anaerob ic d iges t ion 387

The response of the digester contents in the mass spectrometer reaction vessel to a volumetric overload is shown in Fig. 3a, in which the flow rate of medium to the digester was increased in- stantaneously at zero time from 0.12 ml h -1 to 0.83 ml h-1 (equivalent to a retention time of 1 day and a COD loading rate of 9.6 Kg (COD) m - ~ day-1). The rapid decline in the rate of me- thanogenesis after an initial stimulation accompa- nied by the evolution of H2 was observed. The pH of the digester dropped to 5.8 after 9 h from its

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Fig. 3. (a) Effects o f vo lumet r i c over load on an uncon t ro l l ed anaerob ic digester. Re ten t ion t ime was dec reased at t ime zero f rom 7 days to 1 day. (b) Effects o f vo lumet r ic over load on a control led anae rob ic digester. At t ime zero the re tent ion t ime was dec reased f rom 7 days to 1 day. The h y d r o g e n - d e p e n d e n t control sys t em was set to swi tch of f the m e d i u m supp ly p u m p at a H: t h r e sho ld o f 1 i.tM

initial level of 7.0. Figure 3b shows the effect of the same potential overload as in Fig. 3a with the Hz-dependent control system set to switch off the medium supply pump at a H2 threshold greater than 1 gM. The CH4 concentration increased from its initial steady state level of 87 p~M to reach a new steady state of 109 gM equivalent to a me- thanogenic rate of 23 gM min -1. The dissolved H2 oscillated around the threshold level as shown in Fig. 3b with a period of approximately 1 rain. The controlled loading rate was about 4.8 Kg (COD) m -3 day -~. No long term adaptation to further increased methanogenesis was observed.

Although propionate oxidation is inhibited by comparatively low (<0.1 ~tM) concentrations of H2 the present work has shown that the biometha- nation process can be maintained at higher H2 concentrations. This apparent discrepancy can be explained by postulating the existence of micro- niches in which the propionate oxidisers are pro- tected from the comparatively high H2 concentra- tions of the common H2 pool by the juxtaposi- tioning of the H2 producers and consumers (me- thanogens) (Conrad et al. 1985). From Fig. 3b tak- ing the mean H2 concentration of 1 gM and using the same methods of calculation as the above au- thors, the percentage of CH4 originating from the measured dissolved H2 was 0.3%, compared with the expected value of 30% (Boone 1982; Mount- fort and Asher 1981). Hence, only 1% of the total CH4 produced would have originated from the common H2 pool. The interspecies H2 transfer rate was calculated at 27 gM min-1. The equival- ent figures determined for sewage sludge from a municiple digester by Conrad et al. (1985) were 5% and 7.5 IxM min -1, respectively. A possible explanation for these differences is that a more structured community might have developed in the laboratory digester which had been main- tained in a steady state for a period of approxi- mately 2 years.

The laboratory-scale experiments described here suggest that control of digester performance based on the concentration of dissolved H2 pro- vides a valuable approach to optimisation of me- thanogenic rates by use of a single process varia- ble. On-line operation of the membrane probe in situ in the digester follows logically from the work reported here, although possible difficulties arise then from slow stirring, consequent incomplete mixing, and long signal delays. Very rapid stirring may disturb microheterogeneity of flocs, and hence synergistic interactions between organisms. Scale-up in well mixed plant seems eminently fea- sible, especially as long-term mass spectrometric

388 T.N. Whitmore et al.: Hydrogen control of anaerobic digestion

monitoring a pilot plant level for antibiotic pro- duction has been described (Lloyd et al. 1985).

Acknowledgement. The financial assistance of the AFRC is gratefully acknowledged.

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Received November 5, 1986/Revised February 9, 1987