Presented in Session PP2A Bioenergy 1 th 11 IWA World Congress on Anaerobic Digestion 23-27 September 2007 Brisbane, Australia

Compost as a Source of Inoculum for the Anaerobic Digestion of Renewable Biomass, allowing short Hydraulic Retention Times
P. Scherer*, O. Schmidt, M. Unbehauen and L. Neumann Hamburg University of Applied Sciences, Lab. Appl. Microbiology, Lohbrgger Kirchstr. 65, 21033 Hamburg, Germany, * Paul.Scherer@rzbd.haw-hamburg.de Abstract The influence of plant litter-compost as inoculum during continuous anaerobic digestion of fodder beet silage (FBS) was studied. Two parallel reactors (Inoc-1/Inoc-2) were inoculated with liquid manure and sewage sludge. Only one of the reactors was inoculated with compost of the hot rot phase during the startup period (Inoc-2). The aimed hydraulic retention time (HRT) was 10 days. This was achieved in a smooth manner increasing the addition of substrate with response to the activity parameters pH, CH4 and gas production, as established by a self-programmed Fuzzy control. After 150 days the reactor inoculated with compost (Inoc-2) reached a HRT of 10d20d. The reactor without compost (Inoc-1) reached only twice as high HRT of 15 to 40d. On reactor day 243d the substrate feed for both reactors was changed from one time to three times a day. After this the compost reactor (Inoc-2) enabled a preset HRT of 10 days, but the HRT of the other reactor (Inoc-1) remained around 10 to 20 days, for a period of 70 days. This HRT could be also decreased to 10 days by additional inoculation of compost on 314d to Inoc-1. The positive effect of compost allowed a very short HRT with even a somewhat higher the gas production (specGPR 0.58 against 0.55 l/[g*day]. Additional batch digestion tests for 27 days with the same substrate confirmed the increased gas yields by added plant litter compost from the hot rot phase. Keywords Anaerobic digestion; biogas; methane; renewable energy; fodder beet silage; compost

Introduction Biomass can biologically be converted to methane through anaerobic digestion processes. Particularly, production of methane through anaerobic digestion of biomass, namely energy crops and organic wastes, would benefit society by providing a clean fuel from renewable feed stocks. There exists various studies about the applications and benefits of anaerobic digestion processes for production of renewable energy from different sources of biomass However, the influence of operational and environmental parameters on the process performance of the anaerobic biogas digester has to be individually determined, for each particular biomass (substrate type) to be used. A critical step for anaerobic digestion is the start up period. An early report included some observations about the beneficial effect of rotted plant litter on anaerobic digestion to cause a change from acidified digestion to alkalinic digestion of sewage sludge (Reichle, 1935). Other literature described high number of methanogens in compost (Derikx et al., 1989; Thummes et al., 2007). Therefore we applied hot rot compost in previous studies obtaining high digestion rates, but we did not proof its necessity besides the parallel use of swine manure and sewage sludge (Scherer et al., 2001; 2003). Therefore it was the starting point for this study to proof now this compost effect with fodder beet silage as sole substrate in labscale fermentors. Materials and Methods
Laboratory scale reactors

For the experimental work two one-stage laboratory reactors (Inoc-1 and Inoc-2) with a volume of 5.7 liters were constructed at the Hamburg University of Applied Sciences. Both reactors and the substrate tank were cooled to 37C over an external water cooling system (flexible tube). On the start up period the reactors were only fed once per day. After this the substrate feed was performed automatically and controlled by a self programmed software (based on the Fuzzy Logic principle). The produced gas yield was determined with a Milligascounter type MGC10 (Ritter GmbH, Bochum Germany). The CH4 content of the biogas produced by the reactors were analyzed with a two channel IR measurement device (Bluesens GmbH, Herten Germany). The process parameters pH, temperature, gas yield and

Presented in Session PP2A Bioenergy 1 th 11 IWA World Congress on Anaerobic Digestion 23-27 September 2007 Brisbane, Australia

the methane content were measured online and implied every 8 hours over the Fuzzy control the hydraulic retention time (HRT) by the feeding volume. The accurate amount of substrate was accomplished by an electronic balance under the substrate tank, the feed reached the reactor over an automatically driven peristaltic pump. In another self constructed reactor series the balance was outside of the substrate tank and only one pump fed three reactors (Scherer and Lehmann, 2004). Before each feeding sequence the substrate tank and the reactors were completely homogenized by a propeller stirrer for 5 min at 250 rpm. As sole substrate fodder beet silage (brand name Kyros, incl. leaves) was used. This naturally fermented silage had a pH around 3.4 and contained large amounts of volatiles especially lactic acid, acetic acid and ethanol (40 50 % of the total organics). Both reactors were inoculated for the start-up period with 1 litre liquid swine manure and 1 litre sewage sludge. Only reactor Inoc-2 was additionally inoculated with a compost suspension from the hot rot phase (1 litre, 13% VS). Instead of this 1 litre anaerobic water was added to reactor Inoc-1. Both reactors were continuously driven for over 8000 hours. All recorded data from the reactors were collected and saved digitally over the LabView software (National Instruments, Austin USA). After 240 days the feeding rate but not the feeding amount was changed from one time to three times a day. On reactor day 314d compost suspension (from the hot rot phase) corresponding to the start inoclum of the reactor Inoc-2 was added to reactor Inoc-1.
C C M Pump

TIR

Storage Container

WIR CH4 Gas Volume QIR QIR C M C

Figure 1 Flow chart of the two laboratory scale biogas reactors Inoc-1 an Inoc-2. Legend:
Q M W T R I C = = = = = = = quality of a measured value motor (electric) weight temperature recorded values Instrument Controller

TIR pH QIR

Biogas Reactor Discharge

Anaerobic batch digestion tests

For the anaerobic batch digestion tests 1 litre glass bottles with a gas tight rubber plug were filled up to a volume of 500 ml and incubated at 37C. The produced gas was measured with the smaller Milligascounter type MGC1 instead of type MGC10 for the reactors (Ritter GmbH, Bochum Germany). The combination of 1% total solids (TS) compost (from the hot rot phase) and 1% TS of sewage sludge was compared with an inoculation of only 2% TS sewage sludge as bacterial inoculum (n=3). Each vessel was supplemented by 40g of fresh substrate (fodder beet silage) and by a mineral and trace element solution to prevent any mineralic deficiencies (Scherer and Kneifel, 1983). The inoculation the vessels were filled up to 500g with demineralised water. Additionally a reference test serial was inoculated with the same amount of compost, sewage sludge and minerals but without fresh substrate (n=3). The gas yield of the reference series was later subtracted from the gas yields of the main test vessels to generate the net gas production. The initial pH value of the different batch test vessels was adjusted to neutral (pH 7.0-7.3) when it was below acidic. The digestion tests were incubated for 27 days and the gas yield was saved online over a self constructed data logger and a self programmed software.

Presented in Session PP2A Bioenergy 1 th 11 IWA World Congress on Anaerobic Digestion 23-27 September 2007 Brisbane, Australia

Figure 2 Incubator with a digestion test series and online registration of the gas yield by Milligascounters type MGC1 (Ritter GmbH, Bochum Germany) for each vessel. Recorded data were saved digitally (personal computer) over a self constructed data logger station. Microscopically investigation of reactor material

The microbial population of the batch tests and the reactor material was investigated with a Leica DMRB epifluorescence microscope by a 640 times magnification. To determine the total bacterial counts the phase contrast modus was used. For methanogenic bacteria fluorescence stimulation was reached with 420 nm (emission 500nm). All samples were separated for 5 minutes in a special, narrow Nissel tube und diluted 10 times with the anti fading substance DAPCO. Images were taken with the digital Leica DC300 digital microscope camera and evaluated with the image analysis software Image Pro Plus 6.0 (Media Cybernetics, Bethesda USA). Material from reactor day 230d, 303d and 351d (n=10) was investigated, calculating the number of methanogens and the total cell count in cells per ml. Results
Laboratory scale reactors

The main aim of the comparative fermentation was the reduction of the Fuzzy controlled hydraulic retention time (HRT) with no decrement of the specific gas production (specGPR). The specGPR represents liter biogas per liter reactor volume and gram volatile solids (volatile suspended solids plus dissolved volatile solids). After a start up period of 160 days both reactors reached stable running conditions, Inoc-1 had a HRT of 40 days and sepcGPR around 0.47 l/(gVS*day) (Fig. 3 A). Reactor Inoc-2 with compost inoculation reached a HRT around 10 days and a specGPR around 0.53 l/(gVS*day) (Fig. 4 A). The fuzzy controlled reactor reacted with an automatic increase of the HRT if the CH4 content, specGPR or pH value of the reactor decreased. The change of the feeding rate to three times a day on reactor day 240d was followed by an instable run of the reactor for approximately 20 days (not shown). After this instable period the HRT of the reactor Inoc-2 remained at 10 days thereby the specGPR increased to values around 0.56 l/(gVS*day) (Fig. 4B). The HRT of reactor Inoc-1 decreased after reactor day 260d to approximately 15 days thereby the specGPR reached 0.53 l/(gVS*day) (Fig. 3B). After the additional inoculation of compost on reactor day 314d the HRT of reactor Inoc-1 further stabilised and levelled to 10 days HRT (Fig. 3C) whereas the specGPR increased to values around 0.54 l/(gVS*day) (Fig. 3C). In reactor Inoc-2 no further variation was applied after reactor day 260d, the HRT levelled to 10 days. Only the specGPR behaved somewhat instable but stayed finally at about 0.56 l/(gVS*day), (Fig. 4C).

Presented in Session PP2A Bioenergy 1 th 11 IWA World Congress on Anaerobic Digestion 23-27 September 2007 Brisbane, Australia

0,60 0,58 0,56 0,54

HRT leveled to ~40d

HRT leveled to ~10-20d

HRT leveled to 10d smooth

90 80 70 60 50

spec. GPR [l/(gVS*day)]

0,52 0,50 0,48 0,46 0,44 0,42 160

After Changing the Feeding Rate from 1x to 3x/day

30 20 10

[d]
170 180 190 260

Reactor Day [d]

270 280 290 300 310

[d]
320

0 330

Figure 3 (A/B/C) Reactor Inoc-1 without compost addition to the start inoculum: A: Protocol of the biogas reactor Inoc-1 after 160 days. B: After 240 days the feeding rate was changed from 1 to 3 times a day. C: On reactor day 314d inoculation with a compost suspension was performed by replacing a part of the fermentor liquor by approximately 1 litre (13 % VS). The compost was plant litter compost of the hot rot phase.

0.66 0.64 0.62 0.60

HRT leveled to ~10-20d

HRT leveled to 10d After Changing the Feeding Rate from 1x to 3x/day

HRT leveled to ~10-15d

90 80 70 60 50 40 30 20 10

Start Up Period (0 - 240d)

No further Variation

specGPR [l/(gVS*day)]

0.58 0.56 0.54 0.52 0.50 0.48 150

[d]
160 170 180

Reactor Day [d]

260 270 280 290 300 310

[d]
320

0 330

Figure 4 (A/B/C) Reactor Inoc-2 plus compost addition right to the start inoculum: A: Protocol after 160 days of the start up period. B: After the change of feeding rate from 1 to 3 times a day. C: In comparison to the same time period of reactor Inoc-1, no additional compost inoculation (Fig.3 C). Anaerobic batch digestion tests

The gas yields of the batch digestion assays with fodder beet silage (FBS) increased within the first day exponential. On the first day both test assays with FBS reached gas yield values between 1000 and 1300 mlSTP. After this starting period both assay types had a stable gas yield. This lag-like second period lasted for the tests with compost 8 days and for the assays without compost 19 days and was followed by a period of intense gas production. On day 22 the assays additionally inoculated with compost reached a gas yield of 3000 mlSTP. That increased only slightly to a final value of 3280 mlSTP on day 27. The digestion assays without compost showed an increased gas yield between day 19 and 24 to a value around 2000 mlSTP. After this period the final gas production revealed only 2100 mlSTP after 27 days. The gas yield of both blank samples remained at values under 300 mlSTP for the whole test series (Fig. 5).

HRT (h)

HRT (d)

Start Up-Period (0d - 240d)

On 314d Compost Addition

40

Presented in Session PP2A Bioenergy 1 th 11 IWA World Congress on Anaerobic Digestion 23-27 September 2007 Brisbane, Australia

4000 3500 3000 Gas yield (mlSTP) 2500 2000 1500 1000 500 0 0 3

2 % TS Sew age Sludge +40 g Fodder Beet Silage 1 % TS Sew age Sludge +1 % TS Compost +40 g Fodder Beet Silage Blank 2% TS Sew age Sludge Blank 1% TS Sew age Sludge and 1% TS Comopst 3280

2100

12

15

18

21

24

27

Days
Figure 4 Gas Yields of anaerobic batch digestion tests with and without compost. Test run for 27 days at 37C. Inoculation with 1% total solids (TS) compost (from the hot rot phase of plant litter) and 1% TS of sewage sludge compared with 2 % TS of sewage sludge as inoculation. STP = standard temperature (0C) and pressure (1 atm), moisture corrected gas (minus 10-15% of the volume). Microscopically investigation of reactor material

The evaluation of the microbial population and therefore of the active biomass showed for both reactors nearly constant values of methanogenic or total bacteria counts. For both reactors the total bacteria counts remained around 1.2 and 1.5*1010 cells/ml and the number of methanogens around 1.0 and 1.8*109 cells/ml (percentage of 12%) (Fig. 5 A/B). Also the morphological analysis of the bacterial population showed no significantly change over the observed period (pictures not shown). According to this the VSS content of the reactors remained nearly constant at 2%.
1,51E+10 1,30E+10 1,20E+10

1,52E+10

1,51E+10

1,34E+10 1,20E+10

1,30E+10

1,28E+10

1,01E+10

5,10E+09
(11.1%) (12.4%) (9.5%) (8.8%) (13.1%) (14.4%) (12.8%) (8.2%)

1,33E+09

1,88E+09

1,43E+09

1,14E+09

1,76E+09 1,73E+09

1,66E+09

1,04E+09

1,00E+08 Inoc-1 230d Inoc-1 302d Inoc-1 313d Inoc-1 351d Inoc-2 230d Inoc-2 302d Inoc-2 313d Inoc-2 351d

Figure 5 (A/B) Bacterial counts of material from the reactor Inoc-1 [A] started without compost addition. On reactor day 314d compost was added. Reactor Inoc-2 [B] started from the beginning with the addition of compost. Total bacterial counts (dark bars) and number of methanogenic cells (bright bars). Percentages of methanogens to the tolat bacterial counts are set in parentheses.

Presented in Session PP2A Bioenergy 1 th 11 IWA World Congress on Anaerobic Digestion 23-27 September 2007 Brisbane, Australia

Discussion The positive effect of partially decomposed plant litter in contrast to freshly fallen plant litter to convert an acidified sludge into a stable alkaline biogas production of sewage sludge was first reported by Reichle (1935). Later on Ksel and Drake (1996) made similar observations about the positive induction of acetoclastic methanogenesis. Classical and molecular investigations revealed remarkable numbers of methanogens in compost, particulary 107 108/g fresh weight. The Archaea belonged to the genus Methanothermobacter and Methanosarcina thermophila (Derikx et al., 1989; Thummes et al., 2007). Conclusions Both experimental test series with continuously run fermentors and with batch digestion tests confirmed the positive effect of compost on anaerobic digestion as first described by Reichle (1935). During fermentor runs the HRT could be hold shorter by addition of compost (HRT 10-20 days) than without the addition (HRT 15-40 days). An additional positive effect resulted by a modified feeding modus of the reactors from 1 time to 3 times a day. The specGPR of the fermentors, but also the gas yields of the batch tests increased. A most pronounced test result is shown in Fig. 4. Other results of batch tests revealed lower effects on the height of the gas yields, but exhibited an accelerated gas production rate (data not shown). Acknowledgements The authors would like to express their gratitude to Nils Sharfenberg and Christian Rsner for their technical support regarding chemical analyzes. The authors would also like to thank Karsten Lehmann for his technical assistance during reactor management. This project was supported by the government, grant BMBF KFZ 03SF 0317 I. References
Derikx, P.J.L., de Jong, G.A., Op den Camp, H.J., van der Drift, C., van Griensven L.J., Vogels G.D. (1989) Isolation and characterization of thermophilic methanogenic bacteria from mushroom compost. FEMS Microbiol. Lett., 62, 251-258. Ksel, K., Drake, HL. (1996) Anaerobic capacities of leaf litter. Appl Environ Microbiol., 62 (11), 4216-4219. Reichle, C. and Sander, P. (1935) Die Verwendung verrotteten Laubes zur Begnstigung der Zersetzung von Abwasserklrschlamm in nicht blicher Weise arbeitenden Schlammfaulrumen und von schwerer zersetzlichen gewerblichen Abwssern. Gesundheits-Ingenieur, 58 (36), 558-559. Scherer, P., Kneifel, H. (1983) Distribution of polyamines in methanogenic bacteria. J Bacteriol., 154, 13151322. Scherer, P.A., Vollmer, G.-R., Fakhouri, T., Martensen, S. (2000 ) Development of a methanogenic process to degrade exhaustively municipal grey waste under thermophilic and hyperthermophilic conditions. Water Sci. Technol., 41, 83-91. Scherer, P.A. (2001) Influence of high solid content on anaerobic degradation tests measured online by a Milligascounter station for biogas. In: Proceedings of the 9th World Congress on Anaerobic Digestion (L. van Velsen, W. Verstraete, Eds.), University of Antwerpen. Scherer, P.A., Dobler, S., Rohardt, S., Loock, R., Buttner, B., Nldeke, P. and Brettschuh, A. (2003). Continuous biogas production from fodder beet silage as sole substrate. Wat. Sci. Technol., 48, 229-233. Scherer, P.A. and Lehmann, K. (2004). Application of an automatic Fuzzy-logic controller to digest anaerobically fodder beet silage at a HRT of 6.5 days and with an OLR of 14 kg VS/(m*d). In Proceedings of the 10th World Congress of Anaerobic Digestion 2004, pp. 72-78, Montreal. Thummes, K., Kmpfer, P. and Jckel U. (2007) Temporal change of composition and potential activity of the thermophilic archaeal community during the composting of organic material. Syst. Appl. Microbiol., in press.