Relationship between Methanogenic Cofactor Content and Maximum Specific Methanogenic Activity of Anaerobic Granular Sludges

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1988, p /88/ $02.00/0 Copyright D 1988, American Society for Microbiology Vol. 54, No. 5 Relationship between Methanogenic Cofactor Content and Maximum Specific Methanogenic Activity of Anaerobic Granular Sludges LEON G. GORRIS, THEO M. DE KOK, BERND M. KROON, CHRIS VAN DER DRIFT, AND GODFRIED D. VOGELS* Department of Microbiology, Faculty of Science, University of Nijmegen, NL-6525 ED Nijmegen, The Netherlands Received 16 November 1987/Accepted 30 January 1988 In this study we investigated whether a relationship exists between the methanogenic activity and the content of specific methanogenic cofactors of granular sludges cultured on different combinations of volatile fatty acids in upflow anaerobic sludge blanket or fluidized-bed reactors. Significant correlations were measured in both cases between the contents of coenzyme F420-2 or methanopterin and the maximum specific methanogenic activities on propionate, butyrate, and hydrogen, but not acetate. For both sludges the content of sarcinapterin appeared to be correlated with methanogenic activities on propionate, butyrate, and acetate, but not hydrogen. Similar correlations were measured with regard to the total content of coenzyme F420-4 and F420-5 in sludges from fluidized-bed reactors. The results indicate that the contents of specific methanogenic cofactors measured in anaerobic granular sludges can be used to estimate the hydrogenotrophic or acetotrophic methanogenic potential of these sludges. The microbial community involved in anaerobic digestion processes in natural habitats as well as in man-made digestion systems is known to be quite complex, comprising hydrolytic, fermentative, acidogenic, and methanogenic bacteria. A method for the direct and specific determination of the biological potential of the individual trophic groups in anaerobic sludges is not yet available. With regard to the methanogenic bacteria, an estimation of the potential of anaerobic sludges to form methane has been proposed (3) on the basis of the content of coenzyme F420, an electron carrier in methanogenesis and cell carbon synthesis (5, 19). Several assays have been used to quantify coenzyme F420 (3, 16, 21). By using a fluorimetric assay originally developed by Delafontaine et al. (3), a positive correlation has been found for a number of digestion systems between the coenzyme F420 content and the specific methanogenic activity (QCH4, expressed as liters of CH4 per gram of volatile suspended solids [VSS] per day) (2, 3, 11; W. J. de Zeeuw, Ph.D. thesis, Agriculture University of Wageningen, Wageningen, The Netherlands, 1984). The parameter which describes this relation was termed the potential methanogenic activity, QCH4(F420), expressed as liters of CH4 per micromole of coenzyme F420 per day (3). However, some discrepancies have been noticed as well. Dolfing and Mulder (4) reported that coenzyme F420 contents of sludges which had been cultured on different carbon sources in upflow anaerobic sludge blanket (UASB) reactors did not correlate with the QCH4 measured on acetate, but only to the QCH4 obtained on formate. Also, the QCH4(F420) was found to vary for individual digestion systems, with variations in solids retention time, wastewater composition, and physiological growth conditions (12, 22). The observed variations were attributed mainly to shifts brought about in the methanogenic population, since it is known that significant differences exist both in QCH4 (4) and in the coenzyme F420 levels (6, 7, 16) of different methanogenic species. These findings have led to the conclusion that the coenzyme F420 content is not unambiguously correlated to total meth- * Corresponding author. anogenic activity, but rather only to hydrogenotrophic methanogenic activity (4, 22). Recently, methanogenic cofactor assays based on reversedphase high-performance liquid chromatography (HPLC) were introduced by van Beelen et al. (16, 17). By use of these assays and refined versions thereof (7), it was found that structurally distinct types of coenzyme F420 and methanopterin (MPT), a C1 carrier specific for methanogens (18), are present in hydrogenotrophic and acetotrophic species. Generally, the hydrogenotrophic species contain MPT and coenzyme F420-2, whereas the acetotrophic species contain sarcinapterin (SPT) and coenzymes F420-4 and F420-5 (2, 4, and 5 indicate the number of glutamate residues in the side chain). An attractive feature of these assays is the possibility of quantifying both trophic types of methanogens separately in anaerobic sludges on the basis of the different cofactors present (L. Gorris, Ph.D. thesis, University of Nijmegen, Nijmegen, The Netherlands, 1987). With the fluorimetric assays mentioned above, no such distinction is possible and all different types of coenzyme F420 are quantified together. Since the total coenzyme F420 content of anaerobic sludges, however, is not proportional to the total methanogenic activity (4, 12, 22), the HPLC assays were used in this study to investigate whether any correlation exists between hydrogenotrophic or acetotrophic methanogenic sludge activity and the cofactor content in hydrogenotrophic or acetotrophic methanogens, respectively MATERIALS AND METHODS Granular sludge samples. Sludge samples were taken in duplicate from two 4-liter UASB reactors, three 5-liter fluidized-bed (FB) reactors, and six FB reactors with volumes ranging from 650 to 900 ml. All reactors were continuously fed a similar artificially prepared wastewater containing acetate, butyrate, and propionate (A, B, and P, respectively, in ratios below) as carbon sources in addition to essential salts, minerals, and vitamins (Gorris et al., Biotechnol. Bioeng., in press). Both UASB reactors had been seeded with granular sludge from a 5,000-m3 UASB digester (AVEBE; de Krim, The Netherlands) treating po-

2 VOL. 54, 1988 POTENTIAL METHANOGENIC ACTIVITY OF ANAEROBIC SLUDGES 1127 TABLE 1. Cofactor contents and maximum specific methanogenic activities measured for the UASB and FB sludges Cofactor concn QCH4 (VSS) (4mol of CH4 g of VSS-' min-')' Digester (plmol of cofactor g of VSS-1)" on following test substrate: MPT SPT F420-2 F420-5,4b Hydrogen Acetate Butyrate Propionate UASB 2.1 ( ) 1.6 ( ) 0.59 ( ) NM" 4.2 ( ) 6.2 ( ) 6.1 ( ) 6.1 ( ) FB 1.1 ( ) 2.6 ( ) 0.13 ( ) ( ) 2.9 ( ) 26.2 ( ) 13.9 ( ) 9.0 ( ) a Values are means of all data obtained in triplicate analyses for each sludge sample; the range is given in parentheses. b Sum of concentrations of coenzymes F420-5 and F ' NM, Not measurable (cofactor content below detection limit). tato wastewater and received the synthetic wastewater containing the fatty acids at an A/B/P ratio of 1.3:1:1 (wt/vol) at a gradually increasing loading rate (0.3 to 1.7 g of VFA-COD g of VSS-1 day-1). These reactors were operated at 37 C and at a hydraulic retention time of 12 h. Five samples were taken from each reactor over a period of 80 days, starting 10 days after seeding. The three 5-liter FB reactors had been provided with mature FB sludge which had been adapted to the synthetic wastewater at an A/B/P ratio of 3:1:1 (wt/vol) and were fed the wastewater with fatty acids at an A/B/P ratio of 3:1:1, 1:3:1, or 1:1:3 (wt/vol) at a loading rate of 2.0 to 3.0 g of VFA-COD g of VSS-1 day-' at 37 C; the hydraulic retention time was 1.5 h. Five samples were taken from every reactor during a period of 95 days from day 25 after startup. The sludges contained in the other FB reactors had been newly grown, with sand as the support material, on wastewaters containing only acetate or with an A/B/P ratio of 3:1:1 (wt/vol). One sample was taken from each reactor about 136 days after startup. Immediately after sampling, the sludges were washed with anaerobic buffer (10 mm K2HPO4/KH2PO4 [ph 8.0]) and placed under N2 (100%). Measurements and analyses. The VSS content of each sludge sample was determined by standard methods (1). The maximum specific methanogenic activities on acetate, butyrate, propiotnate, or H2-CO2, termed the QCH (VSS), of every sample were assessed by using standardlzed batch activity tests adopted from those of Dolfing and Mulder (4) as described in detail elsewhere (Gorris et al., in press). The concentrations (micromoles of cofactor per gram of VSS) of MPT, SPT, and coenzymes F420-2, F420-4, and F420-5 in the sludge samples were measured by two different binary HPLC assays as described previously (7). The various cofactors were extracted from boiled sludge samples with hot ethanol. MPT and SPT were quantified with an HPLC TABLE 2. (no. 1084B; Hewlett-Packard Co., Palo Alto, Calif.) equipped with a reversed-phase analytical column (0.46 by 10 cm) containing 5 I'm of C18 LiChrosorb RP-18 (E. Merck AG, Darmstadt, Federal Republic of Germany) and with a UV detector at 250 nm. The coenzyme F420 derivatives were analyzed by using an HPLC, composed of M6000 and M45 pumps and a 660 programmer (Waters Associates, Inc., Milford, Mass.), which was equipped with an analytical column (0.46 by 25 cm) packed with 10 I'm of C18 LiChrosorb RP-18. In this case, an Aminco-Bowman spectrophotofluorimeter with excitation and emission wavelengths of 405 and 470 nm, respectively, was the detector. From the data obtained, the potential methanogenic activity relative to the amount of cofactor present, QCH4(cofactor) (expressed as liters of CH4 per micromole of cofactor per day), was derived for each combination of test substrate and cofactor. RESULTS The ranges of data obtained in analyzing the various UASB and FB sludge samples for methanogenic cofactor content and maximum specific methanogenic activities [QCH4(VSS)I on different carbon sources are given in Table 1. The extraction procedure used did not give rise to any other coenzyme F420 derivatives but the types specified; 7-methylpterin, a possible degradation product of MPT analogs (20), was observed in some cases, but accounted for less than 5% of the total content of MPT or SPT (data not shown). With respect to the cofactor contents, smaller amounts of SPT but larger amounts of coenzyme F420-2 were measured in the UASB sludges than in the FB sludges. The MPT contents recorded were within the same range in both sludges, although the average MPT content was higher in the Potential methanogenic activities derived from cofactor concentrations and maximum specific methanogenic activities measured Potential methanogenic activity' (liters of CH4 j.mol of cofactor-1 day-') on following test substrate: Cofactor Digester Acetate Butyrate Propionate H2-C02 SPT UASB 0.13 (0.93), 0.14 (15%) 0.12 (0.72), 0.14 (31%) 0.08 (0.83), 0.14 (21%) 0.10 (0.29), 0.08 (54%) FB 0.35 (0.92), 0.35 (13%) 0.17 (0.81), 0.21 (23%) 0.09 (0.89), 0.14 (15%) 0.03 (0.23), 0.04 (58%) F420-5, -4b FB 104 (0.90), 131 (17%) 42 (0.92), 53 (15%) 27 (0.91), 30 (16%) 37 (0.09), 10 (71%) MPT UASB 0.05 (0.38), 0.09 (50%) 0.07 (0.87), 0.10 (22%) 0.06 (0.89), 0.10 (18%) 0.09 (0.82), 0.12 (26%) FB 0.10 (0.43), 0.88 (51%) 0.17 (0.88), 0.63 (19%) 0.15 (0.86), 0.27 (22%) 0.07 (0.87), 0.10 (20%) F420-2 UASB 0.16 (0.55), 0.35 (44%) 0.25 (0.86), 0.33 (19%) 0.31 (0.86), 0.39 (16%) 0.26 (0.74), 0.19 (30%) FB 1.22 (0.43), 8.47 (33%) 2.38 (0.84), 4.59 (23%) 1.70 (0.92), 3.27 (15%) 0.69 (0.90), 0.78 (16%) a Data are based on 10 and 21 datum points for UASB and FB sludge, respectively. The first value is the slope of a linear regression plot for the curve of QCH4(VSS) versus cofactor content; the correlation coefficient r is given in parentheses. The second value is the average of QCH4(cofactor) values of all samples; the percent standard deviation is given in parentheses. b Coenzymes F420-5 and F420-4 are taken together.

3 1128 GORRIS ET AL. APPL. ENVIRON. MICROBIOL. u,v 30 EU E 20 _ E 110 EU r z0, Om0 sarcinapterin In mol /g V!,SS) FIG. 1. Relation between QCH4(VSS) on acetate and (a) the SPT coenzymes F420-5 and F420-4 of FB sludge. UASB sludges. Coenzymes F420-5 and F420-4 were detectable only in the FB sludges; these coenzymes were quantified together because both types are present simultaneously in acetotrophic series (7, 16). These differences in cofactor contents indicated that the UASB sludges contained more hydrogenotrophic and fewer acetotrophic methanogens than the FB sludges did. This finding was consistent with results obtained by examination of both sludge types with light and epifluorescence microscopes (data not shown). Microscopic examination also indicated that in both sludge types the predominant organisms were Methanothrix spp. Small numbers of Methanosarcina spp. were observed in both cases as well. Identification of these acetotrophic methanogens was based on their characteristic morphology. The values recorded for QCH4(VSS) indicated considerable differences between the biological activities of the two sludge types (Table 1). On acetate, butyrate, or propionate 6 r X090 D~~~~~~~~~~~~~~ B 10 sum of coenzymes F420-5 and L Inmol/g VSS) contents of FB (-) or UASB (0) sludges, and (b) the total content of as the test substrate, QCH4(VSS) values for FB sludge samples were higher than those for UASB sludge samples. The higher activities may have been due to the comparatively larger number of acetotrophic methanogens in the FB sludges. From the QCH4(VSS) found with each test substrate and the concentrations of cofactors measured in each sample, the QCH4(cofactor) for each substrate was assessed in two different ways. First, this parameter was taken as the slope of the linear regression plot for all sample points in the graph of QCH4(VSS) versus cofactor concentration. Second, it was calculated as being the average of the QCH4(cofactor) values of all individual sample points. The data obtained are summarized in Table 2. In most instances, the absolute values of QCH4(cofactor) calculated by either method were comparable. The results show that with acetate as the test substrate, a good correlation was found between QCH4(VSS) and the C-' o.e 4 _0 E, "'-2 / E / E FIG coenzyme F420-2 (nmol /g VSS) methanopterin In mol /g VSS) Relation between QCH4(VSS) of FB sludge on hydrogen and (a) the coenzyme F420-2 content and (b) the MPT content.

4 VOL. 54, 1988 POTENTIAL METHANOGENIC ACTIVITY OF ANAEROBIC SLUDGES 1129 TABLE 3. Potential methanogenic activities of some methanogenic species grown on acetate, calculated from literature dataa on QCH4(VSS) and cofactor contents measured by HPLCb in pure bacterial cultures QCH4(VSS) Cofactor content QCH4 (cofactor) (liters of CH4,umol Species (liters of CH4 g of VSS-' day-') (,umol g of VSS-') of cofactor-' day-') (reference) SPT F420-5,4 QCH4(SPT) QCH4(F420-5,4) Methanothrix soehngenii 1.0 (8) Methanosarcina barkeri MS 2.5 (9) Methanosarcina barkeri Fusaro 1.2 (13) a Data obtained from references cited in parentheses were converted to this uniform dimension if necessary by assuming the following weight ratios: dry/wet, 0.2; protein/dry, 0.5; VSS/dry, (4, 10). b Gorris, Ph.D. thesis. content of SPT, both in UASB sludge and in FB sludge. This is also illustrated in Fig. la. For the FB sludge, the methanogenic activity on acetate appeared to be correlated well with the total content of coenzymes F420-5 and F420-4 (Fig. lb). However, in both sludge types, QCH4(VSS) values measured on acetate were not correlated significantly with the contents of either coenzyme F420-2 or MPT. For hydrogen, the methanogenic activities of the FB sludge appeared to be correlated more with coenzyme F420-2 (Fig. 2a) and MPT contents (Fig. 2b) than with the content of SPT or the total content of coenzymes F420-5 and F420-4 (coenzymes F420-5,4). Also, for the UASB sludges and hydrogen as the test substrate, the correlation of the methanogenic activity with the content of coenzyme F420-2 or MPT was better than with the SPT content. For both butyrate and propionate, significant correlations were found in all cases between QCH4(VSS) and cofactor content. DISCUSSION The results presented here show that the concentrations of specific methanogenic cofactors measured in UASB or FB sludge are indicative of the potential hydrogenotrophic or acetotrophic activity. The contents of MPT and coenzyme F420-2 appeared to be correlated with the hydrogenotrophic methanogenic activity, whereas the contents of SPT and coenzymes F420-5,4 were proportional to the acetotrophic activity (Table 2). In most instances, the absolute values of QCH4(cofactor) derived from linear regression slopes were close to the average values. This would be expected for hydrogen and acetate, which are direct methanogenic substrates. However, with butyrate or propionate as the test substrate, the rate of methane production was dependent in part on the metabolic activity of the acetogenic population, whereas the methane produced in the activity test originated from both acetate and hydrogen, which are intermediates in the degradation of butyrate and propionate to methane. In these cases, the total methanogenic activity, and not the net hydrogenotrophic or acetotrophic methanogenic activity of the sludges, is compared with the contents of cofactors specifically present in hydrogenotrophic or acetotrophic methanogens. Consequently, the average value Of QCH4 (cofactor) is not a direct reflection of the individual methanogenic activities. In contrast, the slope does reflect the net increase in hydrogenotrophic or acetotrophic methanogenic activity per unit of cofactor and thus is the best measure of the potential methanogenic activity of either hydrogenotrophic or acetotrophic methanogens with substrates other than hydrogen or acetate. Tables 3 and 4 list QCH4(cofactor) values calculated for pure cultures of a number of mesophilic methanogenic species. These data show that there are substantial differences both in cofactor contents and in methanogenic activity of the different acetotrophic or hydrogenotrophic species. The QCH4(cofactor) values calculated are also at variance, especially with regard to the two acetotrophic species. The QCH4(SPT) and QCH4(F42o-5,4) values of the FB sludges on acetate (Table 2) were in the range of values for a pure culture of Methanothrix soehngenii. The QCH4(SPT) of the UASB sludges, however, was significantly lower, although microscopic examination had indicated that both types of sludge consisted mainly of Methanothrix-like organisms. The latter inconsistency may have been due to an influence of culture conditions on the acetotrophic activity or to the presence of a different Methanothrix strain in the UASB sludges. In all cases, the QCH4(SPT) and QCH4(F420-5,4) values with butyrate or propionate were lower than the values with acetate. These lower acetotrophic methanogenic potentials may indicate a low convertibility of butyrate and propionate. The QCH4(MPT) and QCH4(F42o-2) values obtained for both sludge types with hydrogen as the test substrate (Table 2) were rather low compared with the values for the hydrogenotrophic methanogens listed in Table 4. These parameters were consistently low with butyrate or propionate as the test TABLE 4. Potential methanogenic activities of hydrogenotrophic methanogens grown in pure culture on H2 C02, calculated from literature data' on QCH4(VSS) and cofactor contents measured by HPLCb QCH4(VSS) Cofactor content QCH4(cofactor) (liters of CH4,mol Species (liters of CH4 g of VSS-' day-') (plmol g of VSS-') of cofactor-' day-') (reference) MPT F420-2 QCH4(MPT) QCH4(F420-2) Methanobacterium formicicum 18.6(15) Methanobacterium bryantii 20.2(14) Methanobrevibacter arboriphilus 12.4(23) Methanospirillum hungatei 10.7(13) "See Table 3, footnote a. b See Table 3, footnote b.

5 1130 GORRIS ET AL. substrate, except for QCH4(F420-2) values for the FB sludges. However, the values of QCH4(F420-2) obtained here are within the range of values which were obtained by Dolfing and Mulder (4) for UASB sludges grown on different combinations of acetate, propionate, and ethanol, namely 0.5 to 2.2 liters of CH4,umol of F420-' day-' (average, 1.4 liters pumol-' day-'). In that investigation, the total content of coenzymes F420 was recorded, by means of a fluorimetric assay, but most of the coenzymes F420 may have been coenzyme F420-2, since the sludges under investigation contained substantial amounts of hydrogenotrophic methanogens, which are characterized by a relatively high content of coenzyme F420-2, in addition to Methanothrix soehngeniilike organisms, which contain only extremely small amounts of coenzymes F420-5 and F420-4 (Table 3). At present, the methane production rate measured in anaerobic digesters is taken as an indication of the biological potential of the methanogenic bacteria present in the sludges. Although this measurement (the actual methanogenic capacity) indicates the methanogenic activity under the prevailing operating conditions, it would be important to establish a measure of the maximum methanogenic capacity, i.e., the activity displayed under ideal operating conditions. Comparisons of maximum and actual methanogenic capacities could then be exploited in the control and optimization of digester performance. The results presented here for two digester systems indicate that a good correlation exists between the content of cofactors characteristic of either hydrogenotrophic or acetotrophic methanogens and the maximum specific methanogenic activity measured on hydrogen or acetate, respectively. In addition, a relationship was found between methanogenic activities on butyrate or propionate and QCH4(cofactor) for each of the various cofactors investigated. Although no single methanogenic cofactor is related to the total maximum methanogenic activity, a valid prediction of acetotrophic or hydrogenotrophic methanogenic potential may, irrespective of the wastewater composition, be obtained by measuring the content of the appropriate specific methanogenic cofactors. ACKNOWLEDGMENT This investigation was supported by the Foundation for Fundamental Biological Research, which is subsidized by the Netherlands Organization for the Advancement of Pure Research. LITERATURE CITED 1. American Public Health Association Standard methods for the examination of water and waste water, 3rd ed. American Public Health Association, New York. 2. Cohen, A., A. M. Breure, D. J. M. Schmedding, R. J. Zoetemeyer, and J. G. van Andel Significance of partial pre-acidification of glucose for methanogenesis in an anaerobic digestion process. Appl. Microbiol. Biotechnol. 21: Delafontaine, M. J., H. P. Naveau, and E.-J. Nyns Fluorimetric monitoring of methanogenesis in anaerobic digestors. Biotechnol. Lett. 1: Dolfing, J., and J. W. Mulder Comparison of methane production rate and coenzyme F420 content of methanogenic consortia in anaerobic granular sludge. Appl. Environ. Micro- APPL. ENVIRON. MICROBIOL. biol. 49: Eirich, L. D., G. D. Vogels, and R. S. Wolfe Proposed structure for coenzyme F420 from Methanobacterium. Biochemistry 17: Eirich, L. D., G. D. Vogels, and R. S. Wolfe Distribution of coenzyme F420 and properties of its hydrolytic fragments. J. Bacteriol. 140: Gorris, L. G. M., and C. van der Drift Methanogenic cofactors in pure cultures of methanogens in relation to substrate utilization, p In H. C. Dubourguier, G. Albagnac, J. Montreuil, C. Romond, P. Sautiere, and J. Guillaume (ed.), Biology of anaerobic bacteria. Elsevier Science Publishers BV., Amsterdam. 8. Huser, B. A., K. Wuhrmann, and A. J. B. Zehnder Methanothrix soehngenii gen. nov. sp. nov., a new acetotrophic non-hydrogen-oxidizing methane bacterium. Arch. Microbiol. 132: Krzycki, J. A., R. H. Wolkin, and J. G. Zeikus Comparison of unitrophic and mixotrophic substrate metabolism by an acetate-adapted strain of Methanosarcina barkeri. J. Bacteriol. 149: Luria, S. L The bacterial protoplasm: composition and organization, p In I. C. Gunsalus and R. Y. Stanier (ed.), The bacteria, vol. 1. Academic Press, Inc., New York. 11. Melchior, J. L., R. Binot, I. A. Perez, H. Naveau, and E.-J. Nyns Biomethanation: its future development and the influence of the physiology of methanogenesis. J. Chem. Technol. Biotechnol. 32: Pause, S. M., and M. S. Switzenbaum An investigation of the use of fluorescence to monitor activity in anaerobic treatment systems. Biotechnol. Lett. 6: Perski, H. J., P. Schonheit, and R. K. Thauer Sodium dependence of methane formation in methanogenic bacteria. FEBS Lett. 143: Roberton, A. M., and R. S. Wolfe Adenosine triphosphate pools in Methanobacterium. J. Bacteriol. 102: Schauer, N. L., and J. G. Ferry Metabolism of formate in Methanobacterium formicicum. J. Bacteriol. 142: van Beelen, P., A. C. DiJkstra, and G. D. Vogels Quantitation of coenzyme F420 in methanogenic sludge by the use of reversed-phase high-performance liquid chromatography and a fluorescence detector. Eur. J. Microbiol. Biotechnol. 18: van Beelen, P., W. J. Geerts, A. Pol, and G. D. Vogels Quantification of coenzymes and related compounds from methanogenic bacteria by high-performance liquid chromatography. Anal. Biochem. 131: van Beelen, P., J. F. A. Labro, J. T. Keltjens, W. J. Geerts, G. D. Vogels, W. H. Laarhoven, W. Guijt, and C. A. G. Haasnoot Derivatives of methanopterin, a coenzyme involved in methanogenesis. Eur. J. Biochem. 139: Vogels, G. D., J. T. Keltjens, T. J. Hutten, and C. van der Drift Coenzymes of methanogenic bacteria. Zentralbl. Bakteriol. Mikrobiol. Hyg. 1 Abt Orig. C 3: White, R. H Methylpterin and 7-methyllumizine: oxidative degradation products of 7-methyl-substituted pteridines in methanogenic bacteria. J. Bacteriol. 162: Whitmore, T. N., S. P. Etheridge, D. A. Stafford, U. E. A. Leroff, and D. Hughes The evaluation of anaerobic digester performance by coenzyme F420 analysis. Biomass 9: Zabranska, J., K. Schneiderova, and M. Dohanyos Relation of coenzyme F420 to the activity of methanogenic microorganisms. Biotechnol. Lett. 7: Zehnder, A. J. B., and K. Wuhrmann Physiology of a Methanobacterium strain AZ. Arch. Microbiol. 111: