The evaluation of anaerobic digester performance by coenzyme F420 analysis

Biomass 9 (1986) 29-35

The Evaluation of Anaerobic Digester Performance by Coenzyme F420Analysis T. N. W h i t m o r e , S. R E t h e r i d g e , D. A. Stafford, U. E. A. Leroff and D. H u g h e s Department of Microbiology,UniversityCollege,Newport Road, Cardiff CF2 1TA, Wales, UK (Received:22 June, 1985) ABSTRACT The performance of pilot plant anaerobic digesters ( plug flow, hydraulic and contact) supplied with pig slurry was assessed in terms of the methane production rate, volatile solids reduction and the methanogenic activity by coenzyme F420 analysis. The plug flow and hydraulic digesters showed higher and more consistent potential methanogenic activities (QcH4(F420)) compared with the contact digester. The performance of the contact digester was also inferior as indicated by volatile solids reduction. Key words: Coenzyme F420 analysis, methanogenic activity, anaerobic digester performance. INTRODUCTION Methanogenesis is an essential step in the process of anaerobic digestion and is often rate limiting, l For better process understanding and control, it is necessary to monitor the activity of methanogenic biomass in anaerobic digesters. The direct enumeration of the strictly anaerobic methanogens by viable counting techniques is both difficult and time consuming, and methods have recently been developed for the estimation of methanogenic biomass. Coenzyme F420, present in all methanogens tested, is a 5-deazaflavin analogue of FMN and functions as a low potential electron carrier, participating in both catabolic and anabolic redox reactions. 2 Because spectrofluorimetric assays of coenzyme F420 are prone to interference effects, 3-5 the partial purification of the coenzyme by ion-exchange chromatography was undertaken in this study, 29 Biornass 0144-4565/86/S03.50 -- © ElsevierApplied SciencePublishers Ltd, England, 1986. Printedin Great Britain

30

T. N. Whitmore, S. P. Etheridge, D. A. Stafford, U. E. A. Leroff, D. Hughes

and the absorbance spectrum was recorded rather than the fluorescence spectrum. This technique was applied to samples taken from different pilot scale anaerobic digesters in order to determine their potential methanogenic activities (QcH4(F420)). This parameter (units :litre CH4 × /~mole- ~coenzyme F420 × day- 1) was proposed by Delafontaine et al. 6 t o measure the methanogenic ability of the microbial community that could be displayed if the environmental conditions were optimal. The performance of these reactors in terms of volatile solids reduction and loading rate was also evaluated, to compare digester performance with methanogenic activity as determined by the coenzyme F420 assay.

Anaerobic digesters The pilot plant anaerobic digesters have been described elsewhere. 7,~ The plugflow digester (30 m 3) was operated at a temperature of 30-35°C and a hydraulic retention time of 15 days. The hydraulic digester (2 m 3) was operated at a temperature of 31-36.5°C and hydraulic retention times between 3 and 5 days. The contact digester (30 m 3) was operated at a temperature of 33.3-38.5°C and a hydraulic retention time of 30 days. Pig waste from a local farm was used as the substrate and samples taken from each digester were always within the range pH 7.5-8.2. A summary of operating parameters is given in Table 1.

TABLE 1

Operating Parameters and Table of Results

Digester

Plugflow Hydraulic Contact

HRT (day)

15 3-5 30

Temperature (° C)

30-35 31-36'5 33-5-38.5

pH

7'9-8'0 7"8-8'2 7.4-7.5

Range of coenzyme F42o concentrations t~mol litre- t

I~mol kg - ~ VS

Specific methanogenic activity +_standard deviation"

0"9-8"0 0"9-4"3 0-09-0.4

20-130 60-170 30-210

0"37_+0'12 0"22 _+0"05 0.01 _+0.06

'~Specific methanogenic activities are the gradients of the lines in Fig. 2. Units are litre ~I=420day- ~.

C H 4 ktmol-

Coenzyme F450analysisof anaerobic digesterperformance

31

The assay of coenzyme F42o

Digester liquor (50 g) was centrifuged (17 000 g, 10 min, 4°C), the pellet was resuspended in 0.5 M NaCI and the centrifuge operation repeated. The pellet was then suspended in water (10 ml) and the cells disrupted by heating in a water bath at 100°C for 10 min. After cooling, the suspension was centrifuged (17 000 g, for 10 min) and the supernatant fluid was diluted 1:2 with 0.3 M NaC1 in 50 n ~ Tris-HC1 buffer (pH 7.5). The solution was then applied to a QAE-Sephadex A-25 column (diameter 1 cm, length 10 cm) according to the method of Schonheit et al., 9 which had been pre-equilibrated with the same buffer. The column was then washed with 20 ml of Tris buffer and the F420 eluted with 1 M NaCI in 50 mM Tris-HCl buffer. The eluate was collected and the absorbance spectrum measured from 350 nm to 450 nm with a Pye Unicam SP1800 dual beam spectrophotometer, against the elution buffer as a blank. From the absorption peak at 420 mn the concentration of F420was determined using an absorption coefficient of 42 500 mol- ~ dm 3 cm- 1.~0Relative standard deviations obtained between multiple assays of identical samples were approximately 10%. The solution exhibited the typical properties of the coenzyme.H The absorption peak disappeared on reduction with dithionite and reappeared on oxygenation and the peak fluorescence emission was recorded at 470 nm when illuminated at 420 nm. Volatile solids (VS) determination

Digester liquor (10 ml) was decanted into a crucible of known weight and placed in an oven at 105°C for 24 h. The crucible and contents were weighed and % total solids (TS) determined. This crucible was then placed in a furnace at 600°C for 30 min and afterwards in a desiccator. The crucible was weighed once again to determine the ash content. The proportion of volatile solids was determined by subtracting the weight of ash from the total solids. Methane determination

The concentration of methane in the digester biogas was determined by use of a GMI Gasco-seeker MK 2 fitted with a pellistor element. The instrument was designed for the detection of methane in air and was recalibrated using standard mixtures of methane in carbon dioxide.

T.N. Whitmore, S. P. Etheridge, D. A. Stafford, U. E. A. Leroff, D. Hughes

32

RESULTS AND DISCUSSION It is common to express methanogen activity in terms of coenzyme F420 concentration in the VS (pmol F420 kg-~ VS) (Fig. 1 ), but when the level of volatile solids present is very low, large errors may result. Therefore F420 levels can also be expressed as volumetric concentrations (pmol F420 litre-1)(Fig. 2). In Fig. 1 it can be seen that there is a high correlation between the rate of methane production and the F420concentration in the volatile solids of both the plugflow (r=0"978) and hydraulic (r=0.999) digesters. The correlation for the contact digester is poor however (r -- 0.411 ) and this is probably due to the low level of volatile solids present which would tend to produce large errors in their measurement. In Fig. 2 the coenzyme F420 concentration and methane production have been related to digester volume. The correlations are good for the plugflow (r= 0.874)and hydraulic digesters (r= 0"978), but the concentration of coenzyme F420 (/,mol litre-') in the contact reactor is comparatively low and the correlation is poor (r--0"142). The low levels of coenzyme F420 and volatile solids present in the contact reactor possibly produced large errors in measurement and it is difficult to draw any conclusions about the relationship between methane production and F420 concentration for this unit.

9O i>,

i

i

i

i

i

I

I

I

I

I '

eo Plugflow

dlge

Plugtlow

•

r =0.978

Hydraulic

0

r =0,999

Contact

•

r =0.411

~ 70 " - ' GO ID er

.I

50

~ 30

~ 20 u

'~ 10 0

I

I

I

40

Coenzyma

Fig. 1.

I

t

8o F420 C .....

t

t

lao

i

leo

i

i

=oo

It-allon Epmolkglvs]

Methane production rate as a function of coenzyme F420 concentration; both parameters expressed in terms of volatile solids.

Coenzyme F45o analysis of anaerobic digester performance I

I

I

I

I

I

I

33

I

0.9 T ~ 0.8 Plugllow

dlges

~ 0.7 Plugflow

•

r = 0.B74

~0.5

tlydraullc

0

r =0.978

,ro o.4

Conlacl

•

r =0.141

g

~ 0.3 o_ 0.2

~ 0.1 i_i_ll ~

act dlgesler I 1

I

I 2

I

I 3

Coenzyme F420ConcentraUon

Fig. 2.

I

I 4

rpmollitrell

Methane production rate per unit volume as a function of coenzyme concentration per unit volume.

F420

The QCH4(F420) values for each digester is represented by the gradients obtained in Fig. 2. These are recorded in Table 1 together with their standard deviation. The difference between the methanogenic potentials may reflect differences in the composition of the methanogen communities in the two digesters. It has been demonstrated that significant differences do exist between the coenzyme F420 levels of different methanogen species. 5,]2 It is also possible that the specific growth rate of methanogens in the hydraulic digester was higher due to the shorter hydraulic retention time, increasing the level of F420 and resulting in a lower specific methane production rate, as demonstrated by Pause and Switzenbaum. ~3 These authors demonstrated decreasing Q)CH4(F42(I) values with decreasing retention times in laboratory scale CSTR digesters. Table 1 shows that the QCH4(F420) values determined for the hydraulic and plugflow digesters are of a comparable magnitude but significantly greater and subject to proportionally less error than the value obtained for the contact digester. The general performance characteristics for each digester have been evaluated and in Fig. 3 the variation of %VS reduction with VS loading rate is shown for each unit. A linear relationship was assumed to exist between these two parameters for each digester type in order to facilitate

T. N. Whitmore, S. P. Etheridge, D. A. Stafford, U. E. A. Leroff, D. Hughes

34

100 90 80 e,~

~

70 ~ ~• , . ~ ~ l l Plugflow

digester

Pluglluw •

r = 0.485

Hydraulic 0

r = 0.543

60

5~ 40

=c 3C 20 0 10

~raulic ~

VS

Fig. 3.

LoadinRat g

digester I

a

I

4

e ['kg n~3da71"]

Performance of the anaerobic digesters expressed as the percentage volatile solids reduction versus the volatile solids loading rate.

comparison between the digester performances. While the correlations are not always good, each data point represents an average value obtained during an extended experimental period (approximately two volume changes). The plugflow digester was only subjected to low loading rates and the VS reduction was always greater than 60%. It is expected that the slope would become more negative at increasing loading rates. The hydraulic digester demonstrated a similar slope, but due to the poor correlation it was difficult to quantify a general trend. The performance of the contact digester was inferior to the plugflow and hydraulic digesters over a similar VS loading range. The correlation was good although only three experimental trials were assessed. At loading rates of approximately 1 kg m -3 day-~ (Fig. 3) the VS reduction for the contact digester was lower than 40% and this rapidly decreased with increasing loading rate. The performance characteristics exhibited by a digester are a function of the microbial population and the engineering designs. The comparatively low QCH4(F420) value for the contact digester, together with its large standard deviation, suggests an unstable or insufficiently acclimatised methanogen population during the experimental period. In addition the digester performance curves (Fig. 3) show that the contact digester was inferior to the hydraulic and plugflow digesters. The estimation of F420 has been criticised for its inability to detect inhibitory effects, since

Coenzyme F45oanalysis of anaerobic digesterperformance

35

inhibited laboratory scale anaerobic digesters have maintained high levels of coenzyme F420.~3 However, since all the digesters were supplied from a c o m m o n source of substrate it is unlikely that toxic substances would have entered solely the contact digester in order to account for its comparatively poor performance characteristics.

ACKNOWLEDGEMENTS The authors gratefully acknowledge the financial support of the E E C , A F R C and the Energy Technology Support Unit, Harwell, during the course of this work.

REFERENCES 1. Archer, D. B. (1983). Enzyme Microb. Technol., 5,162-70. 2. Anthony, C. (1982). The biochemistry of methylotrophs, Academic Press, London, pp. 308-11. 3. Nyns, E. J., Naveau, H. P. & Binot, P. A. ( 1981 ). Biotechnol. Lett., 3,623-8. 4. Hutschemakers, J., Delafontaine, M., Naveau, H. P. & Nyns, E. J. (1982). Biomass, 2,115-25. 5. Van Beelen, P., Dijkstra, A. C. & Vogels, G. D. (1983). Eur. J. Appl. Microbiol. Biotechnol., 18, 67-9. 6. Delafontaine, M. J., Naveau, H. P. & Nyns, E. J. (1979). Biotechnol. Lett., 1, 71-4. 7. Stafford, D. A. & Etheridge, S. P. (1982). Anaerobic digestion, D. E. Hughes et al. (eds), Elsevier Biomedical Press, Amsterdam, pp. 255-68. 8. Etheridge, S. P. (1983). Ind. Biotechnol. Wales, 3, 2-3. 9. Schonheit, P., Keweioh, H. & Thauer, R. K. ( 1981 ). FEMS Microbiol. Lett., 12,347-9. 10. Shauer, N. L. & Ferry, J. G. (1982). J. BacterioL, 150, 1-7. 11. Cheeseman, P., Toms-Wood, A. & Wolfe, R. S. (1972). J. Bacteriol., 112, 527-31. 12. Eirich, L. D., Vogels, G. D. & Wolfe, R. S. (1979). J. Bacteriol., 140, 20-7. 13. Pause, S. M. & Switzenbaum, M. S. (1984). Biotechnol. Lett., 6, 77-80.