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Performance and behaviour of the microbial community of an anaerobic biogas digester using sugar beet silage as mono-substrate
biosystems engineering 102 (2009) 444–452
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/issn/15375110
Research Paper: SEdStructures and Environment
Performance and behaviour of the microbial community of an anaerobic biogas digester using sugar beet silage as mono-substrate Burak Demirela,b,*, Serhat Erguna, Lukas Neumanna, Paul Scherera a
Hamburg University of Applied Sciences (HAW Hamburg), Laboratory for Applied Microbiology, Lohbru¨gger Kirchstrasse 65, 21033 Hamburg, Germany b Bogazici University, Institute of Environmental Sciences, Bebek, 34342 Istanbul, Turkey
article info The performance and the behaviour of the microbial community of an anaerobic mesoArticle history:
philic biogas digester fed with the sugar beet silage (SBS) were investigated in this labo-
Received 1 August 2008
ratory-scale work. The biogas digester was operated using a fuzzy logic control (FLC)
Received in revised form
technique, which was developed at the Hamburg University of Applied Sciences. The SBS,
29 December 2008
which had extremely low pH of 3.3–3.4, was used as mono-input, without addition of
Accepted 27 January 2009
manure. The main objective of this study was to achieve and maintain a stable and safe
Published online 20 February 2009
anaerobic conversion process, along with satisfactory process efficiency. With use of FLC, the biogas digester could be operated with hydraulic retention times (HRTs) in the range 8– 25 days, and up to an organic loading rate (OLR) of 7.41 g volatile solids [VS] l1 d1. The average levels of the specific gas production (GP) rate (SGPR) and the volumetric GP rate (VGPR) were 0.55 l g [VS]1 d1 and 3 l l1 d1, respectively, with 66% of methane (CH4) content in digester biogas. Despite the SBS having an extremely low pH, low amounts of nutrients and buffering capacity, instability problems were not encountered during the experimental period of 156 days, as indicated by the neutral range of the digester pH and the high OLR applied. A daily addition of 1 M KHCO3 to the digester also provided adequate buffering capacity. The application of the FLC also exhibited a positive effect on the methanogenic population. After starting the FLC, the methanogenic population doubled from 4.80 108 to 1.19 109 cells ml1 at the end of the investigation period, whereas the number of the total bacteria remained constant around 2.27–2.30 1010 cells ml1. The FLC application appeared to have a positive effect on the number of the fluorescent methanogens. However, no significant effect on the morphology was found. ª 2009 IAgrE. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
In Germany, following the introduction in 2000 of the Renewable Energy Act (EEG) the anaerobic digestion of
agricultural products (energy crops) and various industrial organic wastes has gained high importance and has received much research attention. In Germany the EEG is the most important driving force for operation of biogas plants for
* Corresponding author. Bogazici University, Institute of Environmental Sciences, Bebek, 34342 Istanbul, Turkey. E-mail address: [email protected] (B. Demirel). 1537-5110/$ – see front matter ª 2009 IAgrE. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biosystemseng.2009.01.008
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biosystems engineering 102 (2009) 444–452
Nomenclature FLC HRT OLR SBS SGPR VFA VGPR VS
fuzzy logic control hydraulic retention time, day organic loading rate, g [VS] l1 d1 sugar beet silage specific gas production rate, l g [VS]1 d1 volatile fatty acid, mg l1 volumetric gas production rate, l l1 d1 volatile solids, g l1
production of renewable energy (mainly electricity) and protection of the climate. The Federal Agricultural Research Centre (FAL) has estimated the total biogas potential in Germany based on available organic wastes, by-products and energy crops, as 24 billion m3 per year (Weiland, 2006). As a result, at the end of 2007, 4000 biogas plants with an overall capacity of 1300 MW were already in operation in Germany (Weiland, 2007). The biological production of biogas from biomass has outstanding advantages. However, in order to profit from anaerobic digestion of biomass (energy crops or any other organic waste), the process should be carefully operated and maintained at optimum operational and environmental conditions. Therefore, the presence, activity and the fate of the microbial community present within the anaerobic biogas digester are also of paramount importance, in addition to the effects of operational and environmental parameters, in order to provide a safe and a stable process for continuous production of methane as a renewable energy source during anaerobic digestion of biomass. Through the implementation of carefully designed ICA (instrumentation, control and automation) strategies for the efficient operation of anaerobic digesters, anaerobic conversion of biomass to biogas can properly be achieved (Steyer et al., 2005). Among these strategies, the fuzzy logic techniques have already been validated as a control strategy for anaerobic wastewater treatment processes (Mu¨ller et al., 1997; Murnleitner et al., 2002; Chen et al., 2003). However, relatively little information is available on the applications of fuzzy logic control (FLC) implemented to operate and monitor the anaerobic digestion of particulate organic wastes and/or energy crops to produce bio-methane. Boscolo et al. (1993) reported the application of fuzzy control for operation of a pilot-scale thermophilic anaerobic digester for the treatment of the organic fraction of municipal solid waste (OFMSW) and the fuzzy logic approach to control anaerobic co-digestion of synthetic pig manure and maize silage were studied by Domnanovich et al. (2003). The continuous anaerobic digestion of an energy crop, namely fodder beet silage (FBS) as mono-substrate, using automatic continuous feeding regulated by the FLC was recently reported by Scherer et al. (2009). In addition, there is also very little available data about the influence of FLC on the fate, behaviour and the activity of the microbial ecology of anaerobic biogas digesters (Scherer et al., 2005). Recent studies focussing on the microbial population dynamics of anaerobic biogas digesters have covered
anaerobic conversion of other substrates like manure, cattle dung and organic fraction of the municipal solid waste, but not with energy crops nor with FLC (Hoffman et al., 2008; Montero et al., 2008; Rastogi et al., 2008; Demirel & Scherer, 2008a). Only a few recent works have investigated the microbial community of an anaerobic biogas digester running on FBS as mono-substrate (Cirne et al., 2007; Klocke et al., 2007). Therefore, the objectives of this experimental work were to investigate the influence of the FLC on the performance of the anaerobic biogas digester fed with a mono-substrate and to investigate the microbial ecology present within the digester with respect to the changes in operational and environmental parameters.
2.
Materials and methods
2.1.
The anaerobic digester and the substrate
A laboratory-scale, single-stage continuous anaerobic digester was used in the experiment. A schematic configuration of the anaerobic biogas reactor is given in Fig. 1. The description and the operation of the anaerobic biogas digester have been previously reported (Demirel et al., 2008; Demirel & Scherer,
Feed
CH4 / CO2
QIR
C
Gas
QIR
M C
Moisture
Temperature
TIR
pH
QIR
Redox
QIR
Effluent
Biogas Reactor 100 mm
Fig. 1 – The scheme of the laboratory-scale anaerobic biogas digester. Q [ Quality of a measured value; M [ motor; T [ temperature; R [ recorded values; I [ instrument; C [ controller.
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2.2.
Table 1 – The general characteristics of the SBS Parameter pH Volatile solids (VS) Ammonium (NHþ 4) Phosphate (PO3 4 ) Acetic acid Propionic acid Isobutyric acid Butyric acid Isovaleric acid Valeric acid Lactic acid Alcohols (methanol, ethanol, propanol; primarily ethanol)
Unit
Value
% mg l1 mg l1 mg l1 mg l1 mg l1 mg l1 mg l1 mg l1 mg l1 %
3.38 19.83 85 583 28 732 3083 294 146 63 70 5600 2.22
The volatile suspended solids (VSS) content was measured according to DIN methods (DIN 38414-8, 1985). Alkalinity, 3 ammonium (NHþ 4 ) and phosphate (PO4 ) were measured according to standard methods (APHA, 1989). Volatile solids (VS) were defined as the sum of the VSS and the volatile dissolved solids (volatile dissolved solids was the sum of volatile fatty acids (VFAs), lactic acid and alcohols). VFA and alcohols were determined using a HP 5890 Series II GC (Agilent) with a flame ionization detector (FID). Lactic acid content was determined enzymatically, using a Merck test (Merck, Darmstadt, Germany).
2.3. 2008b). The digester was operated at a temperature of 41– 42 C. According to Lindorfer et al. (2006), anaerobic digesters fed with energy crops should be operated at temperatures >41 C, as most of the biogas plants have temperatures of 38– 45 C in summer. The reactor was manually fed once a day, with a sugar beet silage (SBS) suspension diluted 1:2 with tap water. Before implementation of FLC, the reactor was operated at a hydraulic retention time (HRT) of 9.5 days, corresponding to a substrate feeding volume of 600 ml d1. After implementation of the FLC, the daily amount of feed was adjusted through FLC. Biogas production was continuously measured online, using a Milligascounter type MGC-10 (Ritter, Bochum, Germany). Methane (CH4) and carbon dioxide (CO2) compositions (v/v) were measured online, using infrared sensors (BlueSens Gas Analyser, Herten, Germany). Temperature, pH and the redox potential (ORP) were also measured continuously online. The SBS (without the leaves or tops) was used as the monosubstrate, without adding manure. The general characteristics of the SBS are given in Table 1. Demirel & Scherer (2008b) provided more detailed information on the characteristics and preparation of the substrate for feeding.
Analytical methods
The fuzzy logic control (FLC)
In order to make the biogas plants more efficient and safe, a controller should be connected to the digester system. One such controller is FLC (Boscolo et al., 1993). The FLC used can be explained by the scheme shown in Fig. 2. The first stage is used to record process measurement values. The second stage is fuzzification, to get a fuzzy set of rules. Fuzzification means that these inputs have to be converted by fuzzification into blurred linguistic terms, since the input parameters are too precise for the human logic (e.g. the linguistic terms high, medium and low can be assumed). The rules of FLC are formulated as a fuzzy set and this is the third stage after fuzzification. The numbers of the rules are determined as 3x, where x represents the input number. The fuzzy logics are based on the human empirically found logic rules (or expert logics). The fourth stage is the defuzzification, this is in order to come to a result. Since FLC provides a conclusion in linguistic terms, the result has to be converted back to a number. The centre of maximum method was chosen and used in this stage (Ross, 2004). Finally, there is a numerical sum as an output in the last stage. This numerical sum can be used by the FLC. Thus, the FLC can determine the feeding volume of a substrate pump of a biogas reactor. The fuzzy
Fig. 2 – The scheme explaining the principle of an FLC for a biogas reactor operated at the Hamburg University of Applied Sciences (HAW Hamburg, Germany). A, B and C are the three selected control parameters, representing the methane content (CH4), pH and the SGPR.
biosystems engineering 102 (2009) 444–452
1.0 0.8
medium
0.4
high range
0.6 low range
0.2 HC 0.0 LC
HC LC
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
Methane (CH4) ranges Fig. 3 – The scheme demonstrating the adjustment of control ranges for the FLC derived substrate feeding according to the measured methane (CH4) percentages in the produced biogas (HC, LC, related to the three ranges of low, medium and high). The medium control range for CH4 percentage was 60–70%. The HC range was defined to be above 75% and below 60% of methane. The LC range is a transient status between a HC and an MR. In the case of methane content, HC value is not achievable, e.g. a high content above 75%.
rules could be programmed and the control range could be adapted to different biogas plants. More details on the applications of FLC for anaerobic digestion of monotype of substrates can be found elsewhere (Scherer et al., 2009).
2.4.
Simulator in Software (SiS)
In order to implement the FLC, a simulator software program was developed (Simulator in Software – SiS, National Instruments, Austin, TX, USA). This software was created using National Instrument Labview (National Instruments, Austin, TX, USA), and it had three input blocks, to enter measurement parameters of CH4, gas production (GP) and pH, and four indicator blocks to show the spec. GP rate (GPR), previous feeding volume, actual feeding volume and the calculated organic loading rate (OLR) percentage. The FLC unit was also built in the SiS, with the help of the PID Control Toolkit from National Instrument.
2.5.
The set of ranges for the sugar beet silage
In addition to the FLC rules, the control ranges of the connected sensors (CH4, pH, spec. GPR) should also be set separately. The importance array of the control parameters should be provided by the FLC rules, but the range settings should additionally be achieved (Scherer et al., 2009). These ranges define the input areas against the linguistic terms and are part of the fuzzy tool of LabView. For example, the low range for the CH4 content in biogas is defined to be between 0 and 60% (Fig. 3). In addition, a high critical (HC) and low critical (LC) range for CH4 should also be set. Thereby, the FLC increases or decreases the dosage of the substrate pump (Figs 2 and 3). The HC range of CH4 usually lies above 75. Values can extend to
447
85%, or even higher (but this is not normally possible). Within the HC range, the FLC should increase the substrate dosage to its preset maximum value, which is related to the OLR of the feeding rate in the previous feeding period. The CH4 values from 0 to 60% can also lie within a HC range, but with an opposite meaning to that above. In this HC range, the substrate feeding should be strongly reduced. The medium ranges (MRs) occur between 60 and 70% CH4, where the OLR remains constant. The LC ranges for the CH4 content in the biogas are transient states between the HC and MR control ranges. In this case, the FLC should slightly decrease the OLR, in relation to the previous substrate dose (Fig. 3). The GPR and pH control ranges have to be selected in a similar manner to the CH4 control. Again they are divided into a HC and LC ranges. The ranges can be changed for every different experiment and the adjustments can be documented, which is easily accomplished by the process computer. More detailed information about the set of ranges that were used for the specific GPR (SGPR) and pH in the FLC for anaerobic digestion of energy crops like sugar or FBSs as mono-substrate can also be found elsewhere (Scherer et al., 2009).
2.6.
Microbiological analyses
The digester material was investigated using a Leica DMRB epifluorescence microscope fitted with a 100 W mercury lamp, by 320 magnification. In order to determine the total bacterial counts, the phase contrast modus was used. For the fluorescent methanogenic population, stimulation was reached with 420 nm (emission 500 nm) (Doddema & Vogels, 1978). All the samples were separated for 5 min in a special narrow Nissel tube (Assistant Glas, Sondheim, Rhoen, Germany) and diluted 10 with anti-fading DAPCO before the microscopic examination (Johansona et al., 1982). All the images were taken with the digital Leica DTC-350Fx digital microscope camera and evaluated with the image analysis software Image Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA).
3.
Results and discussion
3.1.
Digester performance
The digester was operated at a fixed HRT of 9.5 days, without using the FLC, before reactor day 2253. The performance of the digester at steady-state conditions between 2206 and 2228 days is briefly summarized in Table 2. At an OLR of 10 kg [VS] m3 d1, the average SGPR was 0.5 l g1 [VS] d1, at a HRT of 9.5 days, between reactor days 2206 and 2228. The pH was about 7.14 and the volumetric GPR (VGPR) ranged around 5 l l1 d1 (Table 2). Reactor data from days 2229 to 2252 are not reported here because, some modifications carried out in the experimental set-up before running FLC. On reactor day 2253, FLC was started to control the process. The anaerobic digester was operated using FLC for a period of 156 days, between reactor days 2253 and 2409. The overall performance of the digester at steady-state conditions for this period is shown in Table 2. The average value of the SGPR was
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biosystems engineering 102 (2009) 444–452
Table 2 – A comparison of reactor performance without (days 2206–2228) and with FLC (days 2253–2409) Reactor days
HRT (day)
OLR (g [VS] l1 d1)
2206–2228 2253–2409
9.5 8–25 (range)
10.00 2.43–7.41 (range)
SGPRa (l g1 [VS] d1)
VGPRa (l l1 d1)
pHa
CH4 (%)
Redoxa (mV)
0.50 0.55
5.00 3.00
7.14 7.13
69 66
299 382
a Average values at steady-state conditions are reported.
25
8
20 6 OLR
15
4 10 2
HRT
5
2
8
7 pH
1
6
SGPR 5
0 2260
0 2260
2280
2300 2320
2340
2360
2380
0 2400
Reactor operation day Fig. 4 – The levels of OLR and HRT during reactor operation period with FLC using mono-substrate.
pH
30
Hydraulic retention time (HRT) (day)
Organic loading rate (OLR) (g [VS] l-1d-1)
10
supplementation of the digester with an external buffering agent provided a safe operation. Another possibility could be a more variable pH operation with FLC, but without external supplementation (Scherer et al., 2009). This will be the focus of experiments in the near future. Maehnert & Linke (2006) also operated a one-stage biogas system, fed manually once a day. They found out that an abrupt inhibition occurred at an OLR of 4.5 kg [VS] m3 d1 for the beet silage under mesophilic conditions. Similar results were also obtained in another work, with a maximum OLR of 4 kg [VS] m3 d1 for sugar beet or fodder (forage) beet silage under mesophilic conditions (Hassan, 2003). During anaerobic digestion of the FBS, which had a low pH ranging from 3.4 to 4.1, a long-term operation at an OLR level of 12 g [VS] l1 d1 (corresponding to a HRT of 7.5 days) was reported to be possible (Scherer et al., 2003). Klocke et al. (2007) recently reported an OLR range of 1.2–2.3 kg [VS] m3 d1 during anaerobic digestion of FBS as mono-substrate. When the performance of the digester was evaluated as a whole in this study, through application of the FLC strategy in mesophilic anaerobic digestion of a mono-substrate, a safe and a stable process has been achieved, despite the SBS was an extremely acidic- and low-buffered biomass type (Demirel & Scherer, 2008a,b). During mesophilic anaerobic digestion of the FBS as the sole substrate, the pH of the reactor effluent was reported to be around 7.8 (Klocke et al., 2007). The pH level was kept in a lower, but neutral range in this work, due to the lower buffering capacity and the lower ammonium (NHþ 4 ) content of
Specific gas production rate (SGPR) (l g-1[VS] d-1)
0.55 l g1 [VS] d1. The average values of pH and methane (CH4) were determined to be 7.13 and 66%, respectively. In Fig. 4, the levels of OLR and HRT are given for reactor operation period with FLC. The OLR ranged from 2.43 to 7.41 g [VS] l1 d1 and the corresponding HRT levels varied from 8 to 25 days. Some gaps between data points indicate where consistent data could not be obtained due to technical problems during the operation. They included clogging of the gas outlet line due to foam formation and clogging of the feeding tubing. In Fig. 5, the SGPR and the digester pH for the entire operation period (2253–2409 days) are shown. The composition of CH4 in digester biogas for the same period is also shown in Fig. 6. The average SGPR level of 0.55 l g1 [VS] d1 appeared to be in agreement with another study reporting anaerobic digestion of a mono-substrate biomass (Scherer et al., 2007). Since the digester was fed manually only once a day, some fluctuations in SGPR levels could be expected. It was recently reported that one feeding cycle per day would cause performance reduction or cessation of methanogenesis in digesters, which were operated at short HRT levels (Bombardiere et al., 2007). No instability was observed in the present experimental study despite the low levels of HRT and high OLR employed, which was indicated by stable pH levels in the digester and a stable production of CH4 composition in digester biogas (Figs. 5 and 6). The digester was operated within the neutral pH range between reactor operation days 2253 and 2409. If the pH value was lower than that of the pH range settings, then FLC reacted quickly. This meant that on the next day, the digester received a lower amount of feed (or OLR). Since the SBS had an extreme low pH value (Table 1), and an insufficient buffering capacity, the digester had to be externally supplied, adding 40 ml of 1 M KHCO3 solution daily. Therefore, a combination of the FLC implementation and regular
2280
2300
2320
2340
2360
2380
2400
Reactor operation day Fig. 5 – The SGPR and pH for the anaerobic biogas digester operating with FLC from days 2253 to 2409 during anaerobic digestion of SBS (pH of 3.4) as mono-input without addition of manure.
biosystems engineering 102 (2009) 444–452
Methane (CH4) composition (%)
100
80
60
Methane
40
20
0 2260
2280
2300
2320
2340
2360
2380
2400
Reactor operation day Fig. 6 – The methane (CH4) composition of the digester biogas between days 2253 and 2409 during anaerobic conversion of an acidic-low-buffered mono-substrate to methane by using the FLC strategy.
the SBS than those of FBS. The concentrations were reported 1 for the FBS and SBS, respectively to be 440 and 71 mg [NHþ 4]l (Scherer et al., 2009). Furthermore, the average CH4 content of the digester biogas (66%, on average) was higher than that of the previously reported results (Cail & Barford, 1985; Scherer et al., 2003; Klocke et al., 2007). The level of alkalinity and the concentration of VFA are given in Fig. 7. The alkalinity ranged from 2234 to 3890 mg [CaCO3-equivalents] l1, with an average value of 3072 mg [CaCO3-equivalents] l1 from 2253 to 2409 days. The concentration of acetic acid was generally measured to be less than 500 mg l1, except for two incidents, while the concentrations of other VFA were lower than 100 mg l1 during the
449
entire period with FLC. When compared with large-scale biogas plants fed with energy crops with VFA values ranging between 3000 and 5000 ppm, these values appeared to be excellent (Scherer, 2007). In addition to pH, the buffering capacity is another important factor during anaerobic digestion of a monosubstrate (Demirel & Scherer, 2008b). In this study, in terms of buffering capacity and the availability of nutrients, the SBS was a poor substrate. As stated previously, in order to maintain an adequate amount of buffering capacity, KHCO3 was used in the experiments, during operation with and without FLC. Before FLC strategy was implemented, the alkalinity level ranged from 2285 to 2580 mg [CaCO3-equivalents] l1, with an average level of 2438 mg [CaCO3-equivalents] l1 (before day 2253). After the FLC strategy was started, the average alkalinity level increased to more than 3000 mg [CaCO3-equivalents] l1 (Fig. 7). Since the same amount of KHCO3 was added daily to the digester, and the loading was adjusted by the FLC (some days the digester received less feed, but it always received the same amount of KHCO3), the levels of alkalinity in the digester eventually increased, enabling a safer process. The ratio of VFA to alkalinity was also kept below 0.5, in order to achieve stable conditions in the digester. The value of 0.5 comes from anaerobic sewage treatment technology and it guarantees that reactor failure will not occur (Gerardi, 2003; Scherer, 2007). Furthermore, there is also an assumption in literature that the anaerobic digestion with acidic substrates having an alkalinity below 6000 mg [CaCO3-equivalents] l1 should not be possible (Speece, 1996). In spite of this, a stable process was achieved by alkalinity levels ranging between 2234 and 3890 mg [CaCO3-equivalents] l1 in this work (Fig. 7).
3.2.
Microbial population
The changes in the morphology of the microbial community of the mesophilic anaerobic biogas digester were monitored during the operation by FLC using epifluorescence
Fig. 7 – The concentration of VFA and the level of alkalinity during mesophilic anaerobic digestion of the SBS as mono-input.
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biosystems engineering 102 (2009) 444–452
Fig. 8 – Fluorescence pictures from the mesophilic biogas digester operated without FLC (A-day 2253) and with FLC (B-day 2288, C-day 2324, D-day 2358, E-day 2387, F-day 2393).
microscopy. In order to make a comparison, the microbial population of the digester before the FLC operation period was also monitored and presented here. In Fig. 8, the fluorescent images of the reactor material taken on reactor days without FLC operation (on day 2253) and with FLC operation (days 2288, 2324, 2358, 2387, 2393) are shown. Before the FLC was implemented, the digester was operated at a fixed HRT of 9.5 days and an OLR of 10 g [VS] l1 d1, with an average pH of 7.14 (Table 2). On day 2253, before the FLC strategy was begun, this sample was analysed. The qualitative microscopic pictures showed that the effect of the FLC could be seen, firstly on reactor day 2288, which was 35 days after the implementation of the FLC. The quantitative variations in total cell count and the methanogenic cells for days 2253–2393 with FLC are displayed in Fig. 9. After installation of the FLC, between reactor days 2253 and 2288, the total cell count remained nearly stable at 2.3 1010 cells ml1. However, the numbers of the fluorescent methanogenic cells decreased slightly. During the rest of the experiment, the total cell count remained further stable, with minor variations between days 2288 and 2393. After day
2288, the numbers of the fluorescent methanogenic cells increased significantly, reaching 1.19 109 cells ml1 on day 2393 (Fig. 9). In terms of changes within the microbial ecology in the biogas digester with respect to the adjusted OLR and HRT (managed by FLC) and pH, no significant morphological change in the microbial population could be observed during the experiment for a period of 156 days (Fig. 8). Before FLC strategy (on day 2253), coccoid (>1 mm) and medium rod like cells (3–10 mm) dominated the methanogenic population. After operation with FLC, coccoid and medium rod like cells further dominated the biogas digester. During thermophilic (at 55 C) anaerobic digestion of manure or mixture of manure and organic industrial wastes in laboratory-scale biogas digesters, Methanosarcina was identified as the dominant methanogen, at a total VFA level of about 950 mg [acetate] l1 (Schmidt et al., 2000). In this study, the digester was operated at a mesophilic temperature, with an energy crop without manure addition. The acetate levels were also much lower in this work. Therefore, it would be difficult
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1,00E+11 2.44E+10
2.30E+10
2.27E+10
1.94E+10
Cells ml-1
1,00E+10
1.19E+09 9.48E+08
1,00E+09
4.80E+08
Total Cell Count Methanogenic Cells
1,00E+08 2253d
2288d
2324d
2358d
2387d
2393d
451
continuously operated for 6 years without further addition of inoculum, it is unlikely that the inoculum influenced the fate and the behaviour of the microbial population. Previous digester operation procedures and histories can be found elsewhere (e.g. Scherer et al., 2003; Demirel & Scherer, 2008b). It was recently reported that minimal mixing improved methane production 12.5%, in comparison to continuous mixing, during thermophilic digestion of manure (Kaparaju et al., 2008). In this work, the digester was mixed intermittently, 5 min before and after each feeding cycle, totally 10 min per day. However, the effects of different mixing strategies were not investigated in this study. Therefore, the effect of mixing on the performance of the digester and on the behaviour of the microbial ecology could not be evaluated.
Reactor Day Fig. 9 – The total and the fluorescent methanogenic cell counts of the biogas digester operated by FLC using the SBS as the mono-substrate.
to compare the findings of both studies. Elsewhere, it was reported (Karakashev et al., 2005) that the biogas digesters operated with manure were dominated by Methanosarcinaceae, with high levels of ammonia and VFA, while the sludge digesters were dominated by Methanosaetaceae, accompanied by low levels of ammonia and VFA. Karakashev et al. (2005) also reported that the concentrations of ammonia and VFA seemed to have the most influence on the dominant methanogens in biogas digesters. The concentrations of VFA and NHþ 4 did not change much during days 2253 and 2409 in this study. Besides, the morphology of the fluorescent methanogens did not change significantly during this investigation period as well (Fig. 8). The increase in the numbers of the fluorescent methanogens during this investigation period however could be attributed to the influence of the FLC (Fig. 9). On the other hand, according to Karakashev et al. (2005), the loading rates of the investigated systems (full-scale biogas digesters operated with manure or sewage sludge) did not appear to affect the dominant methanogens. In this study, the highest cell counts of the fluorescent methanogens were observed on days 2324 and 2393 (Fig. 8, 9), accompanied by an SGPR level of 0.60 l g1 [VS] d1 (on day 2324) and 0.58 l g1 [VS] d1 (on day 2393). These SGPR levels were obtained at relatively high OLR levels set by the FLC, which in this study were also accompanied by high numbers of methanogens. Therefore, the organic loading seemed to affect the performance of the biogas digester and the methanogenic population significantly during mesophilic anaerobic digestion of the SBS as the sole substrate. During thermophilic-dry anaerobic digestion of the OFMSW, the relative abundance of Archaea and acetoclastic methanogens was reported to directly correlate with OLR, VS removal and CH4 production, which agree with the findings of this study as well (Montero et al., 2008). The fate and behaviour of the methanogenic community correlated closely with the digester performance parameters in our study. In addition, the seed sludge (inoculum) was reported to have no influence on the eventual microbial population (Karakashev et al., 2005). In this work, since the digester had
4.
Conclusions
The implementation of the FLC strategy for mesophilic anaerobic digestion of a mono-substrate (namely the SBS) to produce biogas as a renewable energy source provided a safe and a stable operation in our experiments. Despite the SBS having an extremely low pH and also contained low amount of nutrients and buffering capacity, no instability or process failure were observed within the system, indicated by low concentrations of VFA, an adequate amount of alkalinity and a high OLR in the biogas digester. The digester pH and biogas CH4 percentage also changed very slightly during the operation. The FLC technique applied enabled a process with a high OLR and a low HRT in an anaerobic biogas digester, i.e. a high throughput anaerobic digestion process. The FLC strategy proved to be a useful and reliable tool with a positive effect on the methanogenic population enabling doubling of the number of methanogens during the investigated period of 156 days. Therefore, it can be stated that the behaviour of the methanogenic community correlated quite well with the reactor performance parameters.
Acknowledgements The authors would like to express their gratitude to Nils Scharfenberg, Christian Ro¨sner, Niklas Krakat, Olaf Schmidt and Monika Unbehauen for their help and support during this study.
references
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Research Paper: SEdStructures and Environment
Performance and behaviour of the microbial community of an anaerobic biogas digester using sugar beet silage as mono-substrate Burak Demirela,b,*, Serhat Erguna, Lukas Neumanna, Paul Scherera a
Hamburg University of Applied Sciences (HAW Hamburg), Laboratory for Applied Microbiology, Lohbru¨gger Kirchstrasse 65, 21033 Hamburg, Germany b Bogazici University, Institute of Environmental Sciences, Bebek, 34342 Istanbul, Turkey
article info The performance and the behaviour of the microbial community of an anaerobic mesoArticle history:
philic biogas digester fed with the sugar beet silage (SBS) were investigated in this labo-
Received 1 August 2008
ratory-scale work. The biogas digester was operated using a fuzzy logic control (FLC)
Received in revised form
technique, which was developed at the Hamburg University of Applied Sciences. The SBS,
29 December 2008
which had extremely low pH of 3.3–3.4, was used as mono-input, without addition of
Accepted 27 January 2009
manure. The main objective of this study was to achieve and maintain a stable and safe
Published online 20 February 2009
anaerobic conversion process, along with satisfactory process efficiency. With use of FLC, the biogas digester could be operated with hydraulic retention times (HRTs) in the range 8– 25 days, and up to an organic loading rate (OLR) of 7.41 g volatile solids [VS] l1 d1. The average levels of the specific gas production (GP) rate (SGPR) and the volumetric GP rate (VGPR) were 0.55 l g [VS]1 d1 and 3 l l1 d1, respectively, with 66% of methane (CH4) content in digester biogas. Despite the SBS having an extremely low pH, low amounts of nutrients and buffering capacity, instability problems were not encountered during the experimental period of 156 days, as indicated by the neutral range of the digester pH and the high OLR applied. A daily addition of 1 M KHCO3 to the digester also provided adequate buffering capacity. The application of the FLC also exhibited a positive effect on the methanogenic population. After starting the FLC, the methanogenic population doubled from 4.80 108 to 1.19 109 cells ml1 at the end of the investigation period, whereas the number of the total bacteria remained constant around 2.27–2.30 1010 cells ml1. The FLC application appeared to have a positive effect on the number of the fluorescent methanogens. However, no significant effect on the morphology was found. ª 2009 IAgrE. Published by Elsevier Ltd. All rights reserved.
1.
Introduction
In Germany, following the introduction in 2000 of the Renewable Energy Act (EEG) the anaerobic digestion of
agricultural products (energy crops) and various industrial organic wastes has gained high importance and has received much research attention. In Germany the EEG is the most important driving force for operation of biogas plants for
* Corresponding author. Bogazici University, Institute of Environmental Sciences, Bebek, 34342 Istanbul, Turkey. E-mail address: [email protected] (B. Demirel). 1537-5110/$ – see front matter ª 2009 IAgrE. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biosystemseng.2009.01.008
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Nomenclature FLC HRT OLR SBS SGPR VFA VGPR VS
fuzzy logic control hydraulic retention time, day organic loading rate, g [VS] l1 d1 sugar beet silage specific gas production rate, l g [VS]1 d1 volatile fatty acid, mg l1 volumetric gas production rate, l l1 d1 volatile solids, g l1
production of renewable energy (mainly electricity) and protection of the climate. The Federal Agricultural Research Centre (FAL) has estimated the total biogas potential in Germany based on available organic wastes, by-products and energy crops, as 24 billion m3 per year (Weiland, 2006). As a result, at the end of 2007, 4000 biogas plants with an overall capacity of 1300 MW were already in operation in Germany (Weiland, 2007). The biological production of biogas from biomass has outstanding advantages. However, in order to profit from anaerobic digestion of biomass (energy crops or any other organic waste), the process should be carefully operated and maintained at optimum operational and environmental conditions. Therefore, the presence, activity and the fate of the microbial community present within the anaerobic biogas digester are also of paramount importance, in addition to the effects of operational and environmental parameters, in order to provide a safe and a stable process for continuous production of methane as a renewable energy source during anaerobic digestion of biomass. Through the implementation of carefully designed ICA (instrumentation, control and automation) strategies for the efficient operation of anaerobic digesters, anaerobic conversion of biomass to biogas can properly be achieved (Steyer et al., 2005). Among these strategies, the fuzzy logic techniques have already been validated as a control strategy for anaerobic wastewater treatment processes (Mu¨ller et al., 1997; Murnleitner et al., 2002; Chen et al., 2003). However, relatively little information is available on the applications of fuzzy logic control (FLC) implemented to operate and monitor the anaerobic digestion of particulate organic wastes and/or energy crops to produce bio-methane. Boscolo et al. (1993) reported the application of fuzzy control for operation of a pilot-scale thermophilic anaerobic digester for the treatment of the organic fraction of municipal solid waste (OFMSW) and the fuzzy logic approach to control anaerobic co-digestion of synthetic pig manure and maize silage were studied by Domnanovich et al. (2003). The continuous anaerobic digestion of an energy crop, namely fodder beet silage (FBS) as mono-substrate, using automatic continuous feeding regulated by the FLC was recently reported by Scherer et al. (2009). In addition, there is also very little available data about the influence of FLC on the fate, behaviour and the activity of the microbial ecology of anaerobic biogas digesters (Scherer et al., 2005). Recent studies focussing on the microbial population dynamics of anaerobic biogas digesters have covered
anaerobic conversion of other substrates like manure, cattle dung and organic fraction of the municipal solid waste, but not with energy crops nor with FLC (Hoffman et al., 2008; Montero et al., 2008; Rastogi et al., 2008; Demirel & Scherer, 2008a). Only a few recent works have investigated the microbial community of an anaerobic biogas digester running on FBS as mono-substrate (Cirne et al., 2007; Klocke et al., 2007). Therefore, the objectives of this experimental work were to investigate the influence of the FLC on the performance of the anaerobic biogas digester fed with a mono-substrate and to investigate the microbial ecology present within the digester with respect to the changes in operational and environmental parameters.
2.
Materials and methods
2.1.
The anaerobic digester and the substrate
A laboratory-scale, single-stage continuous anaerobic digester was used in the experiment. A schematic configuration of the anaerobic biogas reactor is given in Fig. 1. The description and the operation of the anaerobic biogas digester have been previously reported (Demirel et al., 2008; Demirel & Scherer,
Feed
CH4 / CO2
QIR
C
Gas
QIR
M C
Moisture
Temperature
TIR
pH
QIR
Redox
QIR
Effluent
Biogas Reactor 100 mm
Fig. 1 – The scheme of the laboratory-scale anaerobic biogas digester. Q [ Quality of a measured value; M [ motor; T [ temperature; R [ recorded values; I [ instrument; C [ controller.
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2.2.
Table 1 – The general characteristics of the SBS Parameter pH Volatile solids (VS) Ammonium (NHþ 4) Phosphate (PO3 4 ) Acetic acid Propionic acid Isobutyric acid Butyric acid Isovaleric acid Valeric acid Lactic acid Alcohols (methanol, ethanol, propanol; primarily ethanol)
Unit
Value
% mg l1 mg l1 mg l1 mg l1 mg l1 mg l1 mg l1 mg l1 mg l1 %
3.38 19.83 85 583 28 732 3083 294 146 63 70 5600 2.22
The volatile suspended solids (VSS) content was measured according to DIN methods (DIN 38414-8, 1985). Alkalinity, 3 ammonium (NHþ 4 ) and phosphate (PO4 ) were measured according to standard methods (APHA, 1989). Volatile solids (VS) were defined as the sum of the VSS and the volatile dissolved solids (volatile dissolved solids was the sum of volatile fatty acids (VFAs), lactic acid and alcohols). VFA and alcohols were determined using a HP 5890 Series II GC (Agilent) with a flame ionization detector (FID). Lactic acid content was determined enzymatically, using a Merck test (Merck, Darmstadt, Germany).
2.3. 2008b). The digester was operated at a temperature of 41– 42 C. According to Lindorfer et al. (2006), anaerobic digesters fed with energy crops should be operated at temperatures >41 C, as most of the biogas plants have temperatures of 38– 45 C in summer. The reactor was manually fed once a day, with a sugar beet silage (SBS) suspension diluted 1:2 with tap water. Before implementation of FLC, the reactor was operated at a hydraulic retention time (HRT) of 9.5 days, corresponding to a substrate feeding volume of 600 ml d1. After implementation of the FLC, the daily amount of feed was adjusted through FLC. Biogas production was continuously measured online, using a Milligascounter type MGC-10 (Ritter, Bochum, Germany). Methane (CH4) and carbon dioxide (CO2) compositions (v/v) were measured online, using infrared sensors (BlueSens Gas Analyser, Herten, Germany). Temperature, pH and the redox potential (ORP) were also measured continuously online. The SBS (without the leaves or tops) was used as the monosubstrate, without adding manure. The general characteristics of the SBS are given in Table 1. Demirel & Scherer (2008b) provided more detailed information on the characteristics and preparation of the substrate for feeding.
Analytical methods
The fuzzy logic control (FLC)
In order to make the biogas plants more efficient and safe, a controller should be connected to the digester system. One such controller is FLC (Boscolo et al., 1993). The FLC used can be explained by the scheme shown in Fig. 2. The first stage is used to record process measurement values. The second stage is fuzzification, to get a fuzzy set of rules. Fuzzification means that these inputs have to be converted by fuzzification into blurred linguistic terms, since the input parameters are too precise for the human logic (e.g. the linguistic terms high, medium and low can be assumed). The rules of FLC are formulated as a fuzzy set and this is the third stage after fuzzification. The numbers of the rules are determined as 3x, where x represents the input number. The fuzzy logics are based on the human empirically found logic rules (or expert logics). The fourth stage is the defuzzification, this is in order to come to a result. Since FLC provides a conclusion in linguistic terms, the result has to be converted back to a number. The centre of maximum method was chosen and used in this stage (Ross, 2004). Finally, there is a numerical sum as an output in the last stage. This numerical sum can be used by the FLC. Thus, the FLC can determine the feeding volume of a substrate pump of a biogas reactor. The fuzzy
Fig. 2 – The scheme explaining the principle of an FLC for a biogas reactor operated at the Hamburg University of Applied Sciences (HAW Hamburg, Germany). A, B and C are the three selected control parameters, representing the methane content (CH4), pH and the SGPR.
biosystems engineering 102 (2009) 444–452
1.0 0.8
medium
0.4
high range
0.6 low range
0.2 HC 0.0 LC
HC LC
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
Methane (CH4) ranges Fig. 3 – The scheme demonstrating the adjustment of control ranges for the FLC derived substrate feeding according to the measured methane (CH4) percentages in the produced biogas (HC, LC, related to the three ranges of low, medium and high). The medium control range for CH4 percentage was 60–70%. The HC range was defined to be above 75% and below 60% of methane. The LC range is a transient status between a HC and an MR. In the case of methane content, HC value is not achievable, e.g. a high content above 75%.
rules could be programmed and the control range could be adapted to different biogas plants. More details on the applications of FLC for anaerobic digestion of monotype of substrates can be found elsewhere (Scherer et al., 2009).
2.4.
Simulator in Software (SiS)
In order to implement the FLC, a simulator software program was developed (Simulator in Software – SiS, National Instruments, Austin, TX, USA). This software was created using National Instrument Labview (National Instruments, Austin, TX, USA), and it had three input blocks, to enter measurement parameters of CH4, gas production (GP) and pH, and four indicator blocks to show the spec. GP rate (GPR), previous feeding volume, actual feeding volume and the calculated organic loading rate (OLR) percentage. The FLC unit was also built in the SiS, with the help of the PID Control Toolkit from National Instrument.
2.5.
The set of ranges for the sugar beet silage
In addition to the FLC rules, the control ranges of the connected sensors (CH4, pH, spec. GPR) should also be set separately. The importance array of the control parameters should be provided by the FLC rules, but the range settings should additionally be achieved (Scherer et al., 2009). These ranges define the input areas against the linguistic terms and are part of the fuzzy tool of LabView. For example, the low range for the CH4 content in biogas is defined to be between 0 and 60% (Fig. 3). In addition, a high critical (HC) and low critical (LC) range for CH4 should also be set. Thereby, the FLC increases or decreases the dosage of the substrate pump (Figs 2 and 3). The HC range of CH4 usually lies above 75. Values can extend to
447
85%, or even higher (but this is not normally possible). Within the HC range, the FLC should increase the substrate dosage to its preset maximum value, which is related to the OLR of the feeding rate in the previous feeding period. The CH4 values from 0 to 60% can also lie within a HC range, but with an opposite meaning to that above. In this HC range, the substrate feeding should be strongly reduced. The medium ranges (MRs) occur between 60 and 70% CH4, where the OLR remains constant. The LC ranges for the CH4 content in the biogas are transient states between the HC and MR control ranges. In this case, the FLC should slightly decrease the OLR, in relation to the previous substrate dose (Fig. 3). The GPR and pH control ranges have to be selected in a similar manner to the CH4 control. Again they are divided into a HC and LC ranges. The ranges can be changed for every different experiment and the adjustments can be documented, which is easily accomplished by the process computer. More detailed information about the set of ranges that were used for the specific GPR (SGPR) and pH in the FLC for anaerobic digestion of energy crops like sugar or FBSs as mono-substrate can also be found elsewhere (Scherer et al., 2009).
2.6.
Microbiological analyses
The digester material was investigated using a Leica DMRB epifluorescence microscope fitted with a 100 W mercury lamp, by 320 magnification. In order to determine the total bacterial counts, the phase contrast modus was used. For the fluorescent methanogenic population, stimulation was reached with 420 nm (emission 500 nm) (Doddema & Vogels, 1978). All the samples were separated for 5 min in a special narrow Nissel tube (Assistant Glas, Sondheim, Rhoen, Germany) and diluted 10 with anti-fading DAPCO before the microscopic examination (Johansona et al., 1982). All the images were taken with the digital Leica DTC-350Fx digital microscope camera and evaluated with the image analysis software Image Pro Plus 6.0 (Media Cybernetics, Bethesda, MD, USA).
3.
Results and discussion
3.1.
Digester performance
The digester was operated at a fixed HRT of 9.5 days, without using the FLC, before reactor day 2253. The performance of the digester at steady-state conditions between 2206 and 2228 days is briefly summarized in Table 2. At an OLR of 10 kg [VS] m3 d1, the average SGPR was 0.5 l g1 [VS] d1, at a HRT of 9.5 days, between reactor days 2206 and 2228. The pH was about 7.14 and the volumetric GPR (VGPR) ranged around 5 l l1 d1 (Table 2). Reactor data from days 2229 to 2252 are not reported here because, some modifications carried out in the experimental set-up before running FLC. On reactor day 2253, FLC was started to control the process. The anaerobic digester was operated using FLC for a period of 156 days, between reactor days 2253 and 2409. The overall performance of the digester at steady-state conditions for this period is shown in Table 2. The average value of the SGPR was
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Table 2 – A comparison of reactor performance without (days 2206–2228) and with FLC (days 2253–2409) Reactor days
HRT (day)
OLR (g [VS] l1 d1)
2206–2228 2253–2409
9.5 8–25 (range)
10.00 2.43–7.41 (range)
SGPRa (l g1 [VS] d1)
VGPRa (l l1 d1)
pHa
CH4 (%)
Redoxa (mV)
0.50 0.55
5.00 3.00
7.14 7.13
69 66
299 382
a Average values at steady-state conditions are reported.
25
8
20 6 OLR
15
4 10 2
HRT
5
2
8
7 pH
1
6
SGPR 5
0 2260
0 2260
2280
2300 2320
2340
2360
2380
0 2400
Reactor operation day Fig. 4 – The levels of OLR and HRT during reactor operation period with FLC using mono-substrate.
pH
30
Hydraulic retention time (HRT) (day)
Organic loading rate (OLR) (g [VS] l-1d-1)
10
supplementation of the digester with an external buffering agent provided a safe operation. Another possibility could be a more variable pH operation with FLC, but without external supplementation (Scherer et al., 2009). This will be the focus of experiments in the near future. Maehnert & Linke (2006) also operated a one-stage biogas system, fed manually once a day. They found out that an abrupt inhibition occurred at an OLR of 4.5 kg [VS] m3 d1 for the beet silage under mesophilic conditions. Similar results were also obtained in another work, with a maximum OLR of 4 kg [VS] m3 d1 for sugar beet or fodder (forage) beet silage under mesophilic conditions (Hassan, 2003). During anaerobic digestion of the FBS, which had a low pH ranging from 3.4 to 4.1, a long-term operation at an OLR level of 12 g [VS] l1 d1 (corresponding to a HRT of 7.5 days) was reported to be possible (Scherer et al., 2003). Klocke et al. (2007) recently reported an OLR range of 1.2–2.3 kg [VS] m3 d1 during anaerobic digestion of FBS as mono-substrate. When the performance of the digester was evaluated as a whole in this study, through application of the FLC strategy in mesophilic anaerobic digestion of a mono-substrate, a safe and a stable process has been achieved, despite the SBS was an extremely acidic- and low-buffered biomass type (Demirel & Scherer, 2008a,b). During mesophilic anaerobic digestion of the FBS as the sole substrate, the pH of the reactor effluent was reported to be around 7.8 (Klocke et al., 2007). The pH level was kept in a lower, but neutral range in this work, due to the lower buffering capacity and the lower ammonium (NHþ 4 ) content of
Specific gas production rate (SGPR) (l g-1[VS] d-1)
0.55 l g1 [VS] d1. The average values of pH and methane (CH4) were determined to be 7.13 and 66%, respectively. In Fig. 4, the levels of OLR and HRT are given for reactor operation period with FLC. The OLR ranged from 2.43 to 7.41 g [VS] l1 d1 and the corresponding HRT levels varied from 8 to 25 days. Some gaps between data points indicate where consistent data could not be obtained due to technical problems during the operation. They included clogging of the gas outlet line due to foam formation and clogging of the feeding tubing. In Fig. 5, the SGPR and the digester pH for the entire operation period (2253–2409 days) are shown. The composition of CH4 in digester biogas for the same period is also shown in Fig. 6. The average SGPR level of 0.55 l g1 [VS] d1 appeared to be in agreement with another study reporting anaerobic digestion of a mono-substrate biomass (Scherer et al., 2007). Since the digester was fed manually only once a day, some fluctuations in SGPR levels could be expected. It was recently reported that one feeding cycle per day would cause performance reduction or cessation of methanogenesis in digesters, which were operated at short HRT levels (Bombardiere et al., 2007). No instability was observed in the present experimental study despite the low levels of HRT and high OLR employed, which was indicated by stable pH levels in the digester and a stable production of CH4 composition in digester biogas (Figs. 5 and 6). The digester was operated within the neutral pH range between reactor operation days 2253 and 2409. If the pH value was lower than that of the pH range settings, then FLC reacted quickly. This meant that on the next day, the digester received a lower amount of feed (or OLR). Since the SBS had an extreme low pH value (Table 1), and an insufficient buffering capacity, the digester had to be externally supplied, adding 40 ml of 1 M KHCO3 solution daily. Therefore, a combination of the FLC implementation and regular
2280
2300
2320
2340
2360
2380
2400
Reactor operation day Fig. 5 – The SGPR and pH for the anaerobic biogas digester operating with FLC from days 2253 to 2409 during anaerobic digestion of SBS (pH of 3.4) as mono-input without addition of manure.
biosystems engineering 102 (2009) 444–452
Methane (CH4) composition (%)
100
80
60
Methane
40
20
0 2260
2280
2300
2320
2340
2360
2380
2400
Reactor operation day Fig. 6 – The methane (CH4) composition of the digester biogas between days 2253 and 2409 during anaerobic conversion of an acidic-low-buffered mono-substrate to methane by using the FLC strategy.
the SBS than those of FBS. The concentrations were reported 1 for the FBS and SBS, respectively to be 440 and 71 mg [NHþ 4]l (Scherer et al., 2009). Furthermore, the average CH4 content of the digester biogas (66%, on average) was higher than that of the previously reported results (Cail & Barford, 1985; Scherer et al., 2003; Klocke et al., 2007). The level of alkalinity and the concentration of VFA are given in Fig. 7. The alkalinity ranged from 2234 to 3890 mg [CaCO3-equivalents] l1, with an average value of 3072 mg [CaCO3-equivalents] l1 from 2253 to 2409 days. The concentration of acetic acid was generally measured to be less than 500 mg l1, except for two incidents, while the concentrations of other VFA were lower than 100 mg l1 during the
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entire period with FLC. When compared with large-scale biogas plants fed with energy crops with VFA values ranging between 3000 and 5000 ppm, these values appeared to be excellent (Scherer, 2007). In addition to pH, the buffering capacity is another important factor during anaerobic digestion of a monosubstrate (Demirel & Scherer, 2008b). In this study, in terms of buffering capacity and the availability of nutrients, the SBS was a poor substrate. As stated previously, in order to maintain an adequate amount of buffering capacity, KHCO3 was used in the experiments, during operation with and without FLC. Before FLC strategy was implemented, the alkalinity level ranged from 2285 to 2580 mg [CaCO3-equivalents] l1, with an average level of 2438 mg [CaCO3-equivalents] l1 (before day 2253). After the FLC strategy was started, the average alkalinity level increased to more than 3000 mg [CaCO3-equivalents] l1 (Fig. 7). Since the same amount of KHCO3 was added daily to the digester, and the loading was adjusted by the FLC (some days the digester received less feed, but it always received the same amount of KHCO3), the levels of alkalinity in the digester eventually increased, enabling a safer process. The ratio of VFA to alkalinity was also kept below 0.5, in order to achieve stable conditions in the digester. The value of 0.5 comes from anaerobic sewage treatment technology and it guarantees that reactor failure will not occur (Gerardi, 2003; Scherer, 2007). Furthermore, there is also an assumption in literature that the anaerobic digestion with acidic substrates having an alkalinity below 6000 mg [CaCO3-equivalents] l1 should not be possible (Speece, 1996). In spite of this, a stable process was achieved by alkalinity levels ranging between 2234 and 3890 mg [CaCO3-equivalents] l1 in this work (Fig. 7).
3.2.
Microbial population
The changes in the morphology of the microbial community of the mesophilic anaerobic biogas digester were monitored during the operation by FLC using epifluorescence
Fig. 7 – The concentration of VFA and the level of alkalinity during mesophilic anaerobic digestion of the SBS as mono-input.
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Fig. 8 – Fluorescence pictures from the mesophilic biogas digester operated without FLC (A-day 2253) and with FLC (B-day 2288, C-day 2324, D-day 2358, E-day 2387, F-day 2393).
microscopy. In order to make a comparison, the microbial population of the digester before the FLC operation period was also monitored and presented here. In Fig. 8, the fluorescent images of the reactor material taken on reactor days without FLC operation (on day 2253) and with FLC operation (days 2288, 2324, 2358, 2387, 2393) are shown. Before the FLC was implemented, the digester was operated at a fixed HRT of 9.5 days and an OLR of 10 g [VS] l1 d1, with an average pH of 7.14 (Table 2). On day 2253, before the FLC strategy was begun, this sample was analysed. The qualitative microscopic pictures showed that the effect of the FLC could be seen, firstly on reactor day 2288, which was 35 days after the implementation of the FLC. The quantitative variations in total cell count and the methanogenic cells for days 2253–2393 with FLC are displayed in Fig. 9. After installation of the FLC, between reactor days 2253 and 2288, the total cell count remained nearly stable at 2.3 1010 cells ml1. However, the numbers of the fluorescent methanogenic cells decreased slightly. During the rest of the experiment, the total cell count remained further stable, with minor variations between days 2288 and 2393. After day
2288, the numbers of the fluorescent methanogenic cells increased significantly, reaching 1.19 109 cells ml1 on day 2393 (Fig. 9). In terms of changes within the microbial ecology in the biogas digester with respect to the adjusted OLR and HRT (managed by FLC) and pH, no significant morphological change in the microbial population could be observed during the experiment for a period of 156 days (Fig. 8). Before FLC strategy (on day 2253), coccoid (>1 mm) and medium rod like cells (3–10 mm) dominated the methanogenic population. After operation with FLC, coccoid and medium rod like cells further dominated the biogas digester. During thermophilic (at 55 C) anaerobic digestion of manure or mixture of manure and organic industrial wastes in laboratory-scale biogas digesters, Methanosarcina was identified as the dominant methanogen, at a total VFA level of about 950 mg [acetate] l1 (Schmidt et al., 2000). In this study, the digester was operated at a mesophilic temperature, with an energy crop without manure addition. The acetate levels were also much lower in this work. Therefore, it would be difficult
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1,00E+11 2.44E+10
2.30E+10
2.27E+10
1.94E+10
Cells ml-1
1,00E+10
1.19E+09 9.48E+08
1,00E+09
4.80E+08
Total Cell Count Methanogenic Cells
1,00E+08 2253d
2288d
2324d
2358d
2387d
2393d
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continuously operated for 6 years without further addition of inoculum, it is unlikely that the inoculum influenced the fate and the behaviour of the microbial population. Previous digester operation procedures and histories can be found elsewhere (e.g. Scherer et al., 2003; Demirel & Scherer, 2008b). It was recently reported that minimal mixing improved methane production 12.5%, in comparison to continuous mixing, during thermophilic digestion of manure (Kaparaju et al., 2008). In this work, the digester was mixed intermittently, 5 min before and after each feeding cycle, totally 10 min per day. However, the effects of different mixing strategies were not investigated in this study. Therefore, the effect of mixing on the performance of the digester and on the behaviour of the microbial ecology could not be evaluated.
Reactor Day Fig. 9 – The total and the fluorescent methanogenic cell counts of the biogas digester operated by FLC using the SBS as the mono-substrate.
to compare the findings of both studies. Elsewhere, it was reported (Karakashev et al., 2005) that the biogas digesters operated with manure were dominated by Methanosarcinaceae, with high levels of ammonia and VFA, while the sludge digesters were dominated by Methanosaetaceae, accompanied by low levels of ammonia and VFA. Karakashev et al. (2005) also reported that the concentrations of ammonia and VFA seemed to have the most influence on the dominant methanogens in biogas digesters. The concentrations of VFA and NHþ 4 did not change much during days 2253 and 2409 in this study. Besides, the morphology of the fluorescent methanogens did not change significantly during this investigation period as well (Fig. 8). The increase in the numbers of the fluorescent methanogens during this investigation period however could be attributed to the influence of the FLC (Fig. 9). On the other hand, according to Karakashev et al. (2005), the loading rates of the investigated systems (full-scale biogas digesters operated with manure or sewage sludge) did not appear to affect the dominant methanogens. In this study, the highest cell counts of the fluorescent methanogens were observed on days 2324 and 2393 (Fig. 8, 9), accompanied by an SGPR level of 0.60 l g1 [VS] d1 (on day 2324) and 0.58 l g1 [VS] d1 (on day 2393). These SGPR levels were obtained at relatively high OLR levels set by the FLC, which in this study were also accompanied by high numbers of methanogens. Therefore, the organic loading seemed to affect the performance of the biogas digester and the methanogenic population significantly during mesophilic anaerobic digestion of the SBS as the sole substrate. During thermophilic-dry anaerobic digestion of the OFMSW, the relative abundance of Archaea and acetoclastic methanogens was reported to directly correlate with OLR, VS removal and CH4 production, which agree with the findings of this study as well (Montero et al., 2008). The fate and behaviour of the methanogenic community correlated closely with the digester performance parameters in our study. In addition, the seed sludge (inoculum) was reported to have no influence on the eventual microbial population (Karakashev et al., 2005). In this work, since the digester had
4.
Conclusions
The implementation of the FLC strategy for mesophilic anaerobic digestion of a mono-substrate (namely the SBS) to produce biogas as a renewable energy source provided a safe and a stable operation in our experiments. Despite the SBS having an extremely low pH and also contained low amount of nutrients and buffering capacity, no instability or process failure were observed within the system, indicated by low concentrations of VFA, an adequate amount of alkalinity and a high OLR in the biogas digester. The digester pH and biogas CH4 percentage also changed very slightly during the operation. The FLC technique applied enabled a process with a high OLR and a low HRT in an anaerobic biogas digester, i.e. a high throughput anaerobic digestion process. The FLC strategy proved to be a useful and reliable tool with a positive effect on the methanogenic population enabling doubling of the number of methanogens during the investigated period of 156 days. Therefore, it can be stated that the behaviour of the methanogenic community correlated quite well with the reactor performance parameters.
Acknowledgements The authors would like to express their gratitude to Nils Scharfenberg, Christian Ro¨sner, Niklas Krakat, Olaf Schmidt and Monika Unbehauen for their help and support during this study.
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