microbial community dynamics of a continuous mesophilic anaerobic biogas digester fed with sugar...

Research Article

Microbial Community Dynamics of aContinuous Mesophilic Anaerobic BiogasDigester Fed with Sugar Beet Silage

The aim of the study was to investigate the long-term fermentation of an extre-mely sour substrate without any addition of manure. In the future, the limitationof manure and therefore the anaerobic digestion of silage with a very lowbuffering capacity will be an increasing general bottleneck for energy productionfrom renewable biomass. During the mesophilic anaerobic digestion of sugar beetsilage (without top and leaves) as the sole substrate (without any addition ofmanure), which had an extreme low pH of around 3.3, the highest specific gasproduction rate (spec. GPR) of 0.72 L/g volatile solids (VS) d was achieved at ahydraulic retention time (HRT) of 25 days compared to an organic loading rate(OLR) of 3.97 g VS/L d at a pH of around 6.80. The methane (CH4) content ofthe digester ranged between 58 and 67 %, with an average of 63 %. The use of anew charge of substrate (a new harvest of the same substrate) with higher phos-phate content improved the performance of the biogas digester significantly. Thechange of the substrate charge also seemed to affect the methanogenic populationdynamics positively, thus improving the reactor performance. Using a new sub-strate charge, a further decrease in the HRT from 25 to 15 days did not influencethe digester performance and did not seem to affect the structure of the methano-genic population significantly. However, a decrease in the HRT affected the size ofthe methanogenic population adversely. The lower spec. GPR of 0.54 L/g VS dattained on day 15 of the HRT could be attributed to a lower size of methano-genic population present in the anaerobic digester during this stage of the pro-cess. Furthermore, since sugar beet silage is a relatively poor substrate, in terms ofthe buffering capacity and the availability of nutrients, an external supply ofbuffering agents and nutrients is a prerequisite for a safe and stable digesteroperation.

Keywords: Anaerobic digestion, Biogas, Biomass, Microbial community

Received: January 31, 2008; revised: May 6, 2008; accepted: May 9, 2008

DOI: 10.1002/elsc.200800010

1 Introduction

The production of biogas, particularly methane (CH4) usingan anaerobic digestion process has gained significant attentionin the last decade. Manure, various types of organic industrialwastes, source sorted household wastes and sewage sludge are

commonly used, along with the energy crops as substrates inbiogas plants for the production of energy. At the end of 2005,3000 biogas plants with an overall capacity of 600 MW werein operation in Germany [1]. Probably, at the end of 2007,nearly 4000 biogas plants with an electrical output of approx.1300 MW would be operating. Furthermore, it can also beassumed that 15 to 20 % of the biogas plants in Germany oper-ate already without any addition of manure [2]. Consequently,an increasing limitation of manure is being observed. There-fore, the recent trend in Germany is the agricultural anaerobicdigestion without any use of manure. Furthermore, the logis-tics to combine meat or milk production with biogas produc-tion is also very difficult.

© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.els-journal.com

B. Demirel1

L. Neumann1

P. Scherer1

1 Hochschule für AngewandteWissenschaften, Fakultät LifeSciences, Lifetec ProcessEngineering, Hamburg,Germany.

–Correspondence: P. Scherer ([email protected]),Hochschule für Angewandte Wissenschaften, Fakultät Life Sciences,Lifetec Process Engineering, Lohbrügger Kirchstrasse 65, D-21033Hamburg, Germany.

390 Eng. Life Sci. 2008, 8, No. 4, 390–398

In addition to the effects of operational and environmentalparameters on the performance of anaerobic biogas digesters,the presence and activity of the microbial community withinthe anaerobic biogas digesters is also of paramount impor-tance, in order to provide a safe and stable process for the con-tinuous production of methane as a renewable energy source.

In the available literature, studies mainly dealt with theinvestigation of the microbial community within an anaerobicbiogas digester. Most of these studies are focusing on thepresence and the activity of the methane forming bacteria dur-ing anaerobic digestion of various types of biomass [3–8],whereas others are focusing on the effects of the environmentalparameters on the microbial population dynamics or the ap-plication of software sensors to monitor the dynamics of themicrobial communities [9–11]. On the other hand, there existslittle information about the bacteriocenosis of anaerobicbiogas digesters operating with energy crops and manure, oreven on anaerobic digesters fed with silage as a mono-input[12–14].

The primary objective of this laboratory-scale study was toinvestigate and monitor the effects of changes in both opera-tional and environmental parameters on the reactor perfor-mance and on the microbial community dynamics during thecontinuous anaerobic digestion of sugar beet silage as the solesubstrate for the production of methane (without the additionof manure). The advantage of smashed and fermented (lacticacid fermentation) silage is that itsharvest can be stored for at least1 year and be quickly converted tomethane. On the other hand, sinceit is fermented so fast and it is ex-tremely acidic, it may cause buffer-ing and foaming problems. Addi-tionally, the sugar beet has gainedinterest as its regulatory price wasdecreased by the EU in 2006, andnow many farmers are looking foran alternative use.

Since there exists little informa-tion about biogas digesters runningon mono-type substrates (whilemost of them are operated as co-digesters, mainly with the additionof manure), the effect of changeson both the operational parametersand the fate and behavior of themicrobial community present inthe lab-scale digester are examined.This study aims to demonstratethat the harvest change of the sametype of an energy crop affects themethanogenic population dy-namics significantly, providing ahigher digester performance, interms of the specific biogas pro-duction rate. However, the effect ofchange in the hydraulic retentiontime on the morphology could beneglected. Besides, the availability

and lack of macro-nutrients (like ammonium) must be initial-ly taken into account by the biogas plant operators as the har-vest of the substrate or the operational parameters (like HRT)are varied during the operation.

Therefore, the preliminary findings of this study would pro-vide biogas plant operators using mono-type substrates (acidicenergy crops that are difficult to handle) valuable informationabout the management of the biogas digester.

2 Materials and Methods

2.1 Description of the Reactor System

A laboratory-scale, single-phase continuous digester was usedin this experimental work for the mesophilic anaerobic diges-tion of biomass. The schematic configuration of the anaerobicbiogas digester is presented in Fig. 1. The reactor was con-structed of glass, and had a total volume of 6 L. The workingvolume was 5.7 L. The reactor was inoculated with 1⁄3 of sew-age sludge, 1⁄3 of swine manure and 1⁄3 of a compost suspensionwithout solids, and has been previously run for more than4 years [15]. The reactor contents were stirred mechanically,using a Heidolph RZR 2020 stirrer (Germany). Temperaturewas kept at 42 °C during the entire operation. Tubes werewrapped around the reactor, and these tubes were connected


Figure 1. Schematic configuration of the anaerobic biogas digester used.

Eng. Life Sci. 2008, 8, No. 4, 390–398 Mesophilic Anaerobic Digestion of Sugar Beet Silage 391

to a water bath (Lauda GmbH, Lauda, Germany), to keep thetemperature constant. There was an overflow tube inside thereactor, which was connected to an effluent tank. The effluenttank was sealed gas tight. The substrate feeding tube and feed-ing ports were located at the top of the reactor. The reactorwas fed once a day, manually, through the substrate feedingport. Gas outlet was located on the top cover of the reactorand connected to a Milligascounter® type MGC10 (Ritter, Bo-chum, Germany), to measure the amount of biogas produceddaily online. There was a condensate trap (a glass bottle of500 mL volume) located in front of the Milligascounter®, tocollect the vapor and to buffer foam expansion. Methane(CH4) and carbon dioxide (CO2) [v/v] levels were measuredonline, using infrared sensors (BlueSens Gaz Analyzer, Herten,Germany). This gas sensor was calibrated by the company,using 16 calibration points and was nearly independent of thegas flow velocity. Before installation, the sensor was calibratedagain using one calibration point. Temperature, pH and redoxpotential (ORP) values were also continuously measuredonline [16].

2.2 Substrate

Sugar beet silage (without top and leaves) was employed as thesingle substrate in this experimental work, without any addi-tion of manure. The general characteristics of the sugar beetsilage are listed in Tab. 1. The substrate was stored at 4 °C untilfurther use. Two harvests of sugar beet silage were used duringthe entire experimental work. Since the sugar beet silage is apoor substrate, in terms of nitrogen (N) and buffering capaci-ty, nitrogen was regularly provided to the feed, by externaladditions of ammonium hydrogen carbonate (NH4HCO3) orammonium chloride (NH4Cl), while sodium hydrogen car-bonate (NaHCO3), potassium hydrogen carbonate (KHCO3)or potassium hydroxide (KOH) were used as buffering agents,in order to keep the reactor pH stable. Stock solutions ofminerals were also prepared and added to the substrate, toprovide phosphate (5.2 mM, Na and K salts), calcium (Ca,1 mM), magnesium (Mg, 2 mM), zinc (Zn, 10 lM), manga-

nese (Mn, 2 lM), copper (Cu, 2 lM), wolfram (W, 1 lM), co-balt (Co, 5 lM), nickel (Ni, 10 lM), selenium (Se, 0.4 lM),molybdenum (Mo, 2 lM), and sulfur (S, 0.5 mM). All chemi-cals were of reagent grade, obtained from commercial sources(Merck, Darmstadt, Germany).

2.3 Analytical Methods

Mixed samples were regularly drawn from the reactor, andmeasured to determine volatile solids (VS), volatile suspendedsolids (VSS), ammonium (NH4

+), phosphate (PO43–), volatile

fatty acids (VFA), alcohols and alkalinity. The VSS content wasmeasured according to the DIN Methods [17]. The alkalinity,ammonium and phosphate were measured according to theStandard Methods [18]. Total VS was defined as the sum ofVSS and volatile dissolved solids (volatile dissolved solids wasthe sum of VFAs and alcohols). Volatile fatty acids (VFA) andalcohols were determined using an HP 5890 Series II GC witha flame ionization detector (FID) and a BP 21 Bonded FFAPFused Silica column. Hydrogen (H2) was used as the carriergas. Injection and detector temperatures were 240 and 260 °C,respectively. 1 lL of sample volume was used for injection.

2.4 Microbiological Analysis

For microbiological investigations, mixed samples were drawnfrom the reactor under steady-state conditions. The steady-state conditions were defined as 3-fold of the HRT. The diges-ter material was investigated using a Leica DMRB epifluores-cence microscope fitted with a 100 W mercury lamp, by a640-time magnification. In order to determine the total bacter-ial counts, a phase contrast modus was used. For the fluores-cent methanogenic population, stimulation was reached with420 nm (emission 500 nm). All the samples were separated for5 minutes in a special narrow Nissel tube (Assistant Glas,Sondheim, Rhoen, Germany) and diluted 10 times with theanti fading substance DAPCO before the microscopic exami-nation [19]. All the images were taken with the digital Leica


Table 1. General characteristics of sugar beet silage.

Parameter Unit Substrate charge-1 Substrate charge-2 Average

pH 3.27 3.38 3.33

Volatile solids (VS) % 18.44 19.83 19.14

Ammonium (NH4+) mg/L 55 85 70

Acetic acid mg/L 13965 28732 21349

Propionic acid mg/L 247 3083 1665

Isobutyric acid mg/L 29 294 162

Butyric acid mg/L 113 146 130

Isovaleric acid mg/L 32 63 48

Valeric acid mg/L 7 70 39

Lactic acid mg/L 11224 5600 8412

392 B. Demirel et al. Eng. Life Sci. 2008, 8, No. 4, 390–398

DC-300 microscope camera andevaluated with the image analysissoftware Image Pro Plus 6.0 (MediaCybernetics, Bethesda USA).

3 Results andDiscussion

An overview of the digester opera-tion and the environmental param-eters under steady-state conditionsis given in Tab. 2. The variations inthe specific (Spec. GPR) and volu-metric (Vol. GPR) gas productionrates are presented in Fig. 2, whilethose in the pH and methane per-centage in the digester are dis-played in Fig. 3. The variations inthe concentrations of ammonium(NH4

+) and phosphate (PO43+) are

shown in Fig. 4.Firstly, substrate charge-1 was

used in our experimental work at25 days of the HRT. The reactorwas operated at an OLR (Organicloading rate) of 3.56 g VS/L d and42 °C. Under steady-state condi-tions on day 25 of the HRT, thespecific and volumetric gas pro-duction rates were determined tobe 0.49 L/g VS d and 1.74 L/L d,respectively, with an average reac-tor pH of 7.27. The methane (CH4) content of the digesterbiogas varied between 51 and 61 % (57 %, on average), and thealkalinity ranged from 5250 to 6825 mg CaCO3/L. The averagealkalinity level was 6032 mg CaCO3/L, while the average con-centrations of ammonium (NH4

+) and phosphate (PO43+) in

the anaerobic biogas digester were 2282 and 48 mg/L, respec-tively. Without ammonium buffering, such an alkalinity levelwas not possible to be maintained in the digester [16].

During this phase of the reactor operation, ammoniumhydrogen carbonate (NH4HCO3) was used daily, in order toprovide sufficient amounts of nitrogen and buffering capacity

in the anaerobic biogas digester, since sugar beet silage (with-out top and leaves) is a relatively poor substrate in terms ofnutrients and buffering capacity. However, as a result of thehigh external addition of NH4HCO3, high concentrations ofNH4

+ and alkalinity were observed in the digester. Therefore,the daily amount of NH4HCO3 used was reduced, in order toprevent a further increase of the NH4

+ levels in the digester(see Fig. 4). In Fig. 5A, the fluorescence picture taken understeady-state conditions on day 25 of the HRT with substratecharge-1 is displayed for the anaerobic biogas digester. Thecounts of the fluorescent methanogens seem quite low during


Table 2. Overview of operational and environmental parameters during the anaerobic digestion of sugar beet silage as a mono-substrate.

Substratecharge(c)

HRT[d]

OLR[g VS/L d]

Temperature[C]

Spec. GPR(a)

[L/g VS d]Vol. GPR(a)

[L/L d]pH(a) Methane(a)

[%]Redox(a)/(b)

[mV]Alkalinity(a)

[mg CaCO3/L]Reactor VSS(a)

[%]

1 25 3.56 42 0.49 1.74 7.27 57 –288 6032 2.65

2 25 3.97 42 0.72 2.86 6.79 63 –272 2115 1.88

2 15 6.33 42 0.54 3.44 7.15 67 –296 3214 1.16

(a) Average values under steady-state conditions of the reactor.(b) Corrected redox potential (ORP) values (–230 mV reference electrode).(c) Substrate charge in this case also means a different harvest.

Figure 2. The specific (Spec. GPR) and volumetric (Vol. GPR) gas production rates during themesophilic anaerobic digestion of sugar beet silage without manure under steady-state condi-tions. As charge-1 ran out, the change to the substrate charge-2, which was naturally rich in phos-phate content, improved the reactor performance substantially.


this phase of the operation, insteadof a 2.65 % of VSS concentrationin the digester. Some coccoidmethanogens and very few metha-nogenic rods could be detected bymicroscopic examinations of thedigester sample. Lower counts ofthe fluorescent methanogens mighthave resulted from the high NH4

+

concentrations externally added(more than 2200 mg NH4

+/L),which could hypothetically have anadverse effect on the presence andactivity of the methanogens [20].However, presumably, even themost ammonia sensitive acetate-utilizing methanogens, the mem-bers of the Methanosarcinaceae,could tolerate high ammonia con-centrations around 4100 mg N/Lin anaerobic biogas reactors [10].The authors have also reportedthat the manure biogas digesterscontained normally high levels ofammonia and VFA concentrations,and were dominated by the mem-bers of the Methanosarcinaceae.Low concentrations of VFA andammonium were observed in ourexperimental work. Furthermore,stable anaerobic digestion of swinemanure for biogas production haspreviously been reported for am-monia concentrations at6000 mg N/L [21]. Therefore, inour case, an adverse effect of highNH4

+ concentrations on themethanogenic population was un-likely to occur.

Since substrate charge-1 ran outduring the experiments, a new sub-strate charge (a new harvest of thesame substrate/substrate charge-2)was later used during 25 days ofHRT operation. Consequently, thereactor output was affected by thisvariation of substrate charge. Dur-ing the operation with substratecharge-2 on day 25 of the HRT, anexternal addition of ammoniumhydrogen carbonate was ceased(see Fig. 4). The objective was tosupply the minimum amount ofNH4

+ required for a stable digesteroperation. Instead, in order to pro-vide the adequate buffering capaci-ty and a stable reactor pH, sodiumhydrogen carbonate (NaHCO3),calcium carbonate (CaCO3) and


Figure 3. The pH and methane percentage in the biogas digester [v/v] during the mesophilicanaerobic digestion of sugar beet silage without manure under steady-state conditions. (SC-1:Substrate charge-1; SC-2: Substrate charge-2). The methane content and pH increased signifi-cantly by the naturally higher buffered substrate charge.

Figure 4. The concentrations of ammonium (NH4+) and phosphate (PO4

3+) during the anaerobicdigestion of sugar beet silage as a mono-substrate without manure. (SC-1: Substrate charge-1;SC-2: Substrate charge-2). Ammonium was artificially supplemented as no manure was used,while the phosphate increase derived naturally from the new harvest of substrate charge-2.


potassium hydroxide (KOH) were tested, in turn, during thisphase of the reactor operation. However, calcium carbonateaddition caused excessive foam formation in the digester,which led to clogging of the gas outline and condensate trap.Sodium carbonate was also avoided later, since sodium ionscould have a positive effect on methanogenesis [22, 23]. Final-ly, an addition of potassium hydroxide (KOH) seemed to func-tion best to keep a stable pH during this phase of operation.

With substrate charge-2, under steady-state conditions for25 days of HRT operation, the specific and volumetric GPRlevels reached 0.72 L/g VS d and 2.86 L/L d, respectively, withan average reactor pH of 6.79. The methane (CH4) content ofdigester biogas varied between 58 and 67 % (63 %, on averagefor substrate charge-2), and the alkalinity ranged from 1580 to2975 mg CaCO3/L (2115 mg CaCO3/L, on average) (seeTab. 2). A slightly higher OLR was used with substratecharge-2 (The OLR was around 3.97 g VS/L d), because sub-strate charge-2 had a higher VS content than that of substratecharge-1 (see Tab. 1). The new charge of substrate provided a

higher volumetric gas production rate and methane content inthe digester biogas produced, due to a higher VS loading. Theaverage concentrations of ammonium (NH4

+) and phosphate(PO4

3+) in the anaerobic biogas digester were 240 and 272 mg/L, respectively. A decrease in the NH4

+ concentration occurreddue to the cease of external addition of ammonium hydrogencarbonate, while higher concentrations of PO4

3+ occurred dueto the use of a new harvest of substrate (substrate charge-2).The PO4

3+ concentration in substrate charge-2 varied between575 and 590 mg/L, while in substrate charge-1, PO4

3+ concen-tration ranged from 2 to 15 mg/L. Since substrate charge-2had a much higher PO4

3+ content than that of the substratecharge-1, higher PO4

3+ concentrations were eventually ob-served in the digester (see Fig. 4). A new charge of substrate,with a much higher PO4

3+ content, seemed to provide a betterreactor performance, particularly in terms of the specific gasproduction rate (Spec. GPR, related to the fed input), onday 25 of the HRT.


Figure 5. (A) Fluorescence picture taken under steady-state conditions from the biogas digester on day 25 of the HRT with substratecharge-1 (silaged sugar beets/harvest-1). (B) Fluorescence picture taken under steady-state conditions from the biogas digester onday 25 of the HRT with substrate charge-2 (silaged sugar beets/harvest-2). (C) Fluorescence picture taken after the reactor failure onday 15 of the HRT with substrate charge-2. (D) Fluorescence picture taken for steady-state conditions from the biogas digester on day 15of the HRT with substrate charge-2. (The HRT periods are shown in Figs 2 and 4).


In Fig. 5B, a typical fluorescence picture taken under steady-state conditions on day 25 of the HRT with substrate charge-2is shown. Methanogenic rods (5–10 lm) (Methanobacteria)seemed to dominate the methanogenic population, along witha few short rods (2 lm), some coccoid methanogens andMethanosarcina. The total cell count was determined to be9.76 × 109, with a fluorescent cell count of 1.81 × 109. The per-centage of the fluorescent methanogens to the total cell countwas approx. 18.6 %. In spite of a relatively lower digester pH(6.8) and VSS content in the digester (1.88 %), in comparisonto the previous run with the old charge of substrate (substratecharge-1), the increased dominance of the fluorescent metha-nogens in the image (compare Fig. 6) was congruently accom-panied with a high specific gas production rate (0.72 L/g VS d)and a higher methane percentage in the biogas digester (63 %).Therefore, the change of the substrate charge seemed to affectthe methanogenic population dynamics positively. Also, thereactor performance was significantly improved, in terms ofthe specific gas production rate on day 25 of the HRT, withthe substrate charge-2. It was earlier reported that the sludgebiogas digesters contained low levels of ammonia and VFA,and were dominated by the members of the coccoid Methano-saetaceae [10]. During this phase of the experiments, low VFAand NH4

+ levels were determined in the digester, too, but nofilamentous Methanosaeta was observed. Perhaps the highOLR influenced the presence of the hydrogenotrophic metha-nogens, but not the low levels of VFA and ammonium [24].

In the following phase of the experiments, the HRT was de-creased from 25 to 15 days. No external ammonium supplywas used on day 15 of the HRT, in order to find out the mini-mum amount of ammonium concentration required for thecontinuous anaerobic conversion of sugar beet silage to meth-ane. It was previously reported that the ammonium concentra-tion had to be kept in excess of at least 40 to 70 mg N/L, toprevent the reduction of bacterial activity [25]. Under meso-philic conditions and at a neutral pH, this means an ammoni-um concentration to be about 10-fold. In a recent paper, it wasalso reported that the optimum growth conditions for Metha-nosaeta concilii were in the range of 250 to 1100 mg NH4

+/L[26]. Potassium hydroxide (KOH) was still used to control thedigester pH. However, within ten days of operation, withoutexternal supply, the ammonium (NH4

+) concentration in thebiogas digester depleted, and the reactor pH declined to 5.5(data not shown). The reactor had previously exhibited a goodperformance in an ammonium concentration range between150 and 77 mg/L at 25 days of the HRT without an externalsupply of ammonium, but it failed at 15 days of the HRT op-eration without an external NH4

+ addition (see Figs. 2 and 4).During this failure period, the maximum concentrations ofacetic, propionic, butyric, valeric, isovaleric and isobutyricacid were measured to be 6238, 3514, 1946, 1170, 971 and114 mg/L, respectively. The fluorescence picture taken fromthe biogas digester just after the failure is displayed in Fig. 5C.There exists some fluorescent methanogens in the digester,however, their activity is decreased, due to high concentrationsof VFAs accumulated within the digester. Mostly acetate-utiliz-ing methanogens seemed to be affected adversely during thefailure. Propionic and butyric acid-utilizing methanogenscould have also been severely affected.

In order to recover the digester, the HRT was firstly adjustedfrom 15 to 100 days, and then to 75, 50 and 25 days, respec-tively. During this recovery phase, firstly ammonium hydrogencarbonate, and then potassium hydroxide were used to provideNH4

+ and buffering capacity to the biogas digester. After stableconditions had been maintained in the digester (it took almost90 days), the HRT was finally adjusted to 15 days again. At15 days of the HRT operation with substrate charge-2, ammo-nium chloride (NH4Cl) and potassium hydrogen carbonate(KHCO3) were both used to provide NH4

+ and bufferingcapacity to the biogas digester. Under steady-state conditionson day 15 of the HRT, the specific and volumetric GPR levelswere determined to be 0.54 L/g VS d and 3.44 L/L d, respec-tively, with an average digester pH of 7.15. The methane com-position of digester biogas varied between 62 and 71 % (67 %,on average). The alkalinity was 3214 mg CaCO3/L (on aver-age). With substrate charge-2 on day 15 of the HRT, the biogasdigester exhibited a lower specific gas production rate, about25 % less (see Tab. 2).

In Fig. 5 D, the fluorescence picture taken under steady-stateconditions on day 15 of the HRT with substrate charge-2 ispresented. Long methanogenic rods seemed to dominate themethanogenic population during this stage of operation, alongwith some coccoid methanogens. A change in the HRT from25 to 15 days did not seem to affect the morphology of themethanogenic population significantly. The total cell countwas determined to be 2.77 × 1010, with a fluorescent cell countof 1.11 × 109. The percentage of the fluorescent methanogensto the total cell count was noted to be approx. 4.0 %. Thechanges in the numbers of the total bacterial count and thefluorescent cells on day 25 and 15 of the HRT with substratecharge-2 are displayed in Fig. 6. With the decrease in the HRTfrom 25 to 15 days, the fluorescent cell count decreased. Thepercentage of the fluorescent cell count to the total cell countalso decreased from 18.6 to 4.0 %. Despite a higher influentfeeding rate on day 15 of the HRT, and without any sludgerecycling, the total cell count increased, with respect to thedecrease in the HRT. A lower specific gas production rate of0.54 L/g VS d on day 15 of the HRT could be attributed to alower amount of the methanogenic population present withinthe anaerobic digester. Therefore, the decrease in the HRTseemed to affect the size of the methanogenic populationadversely, resulting in a relatively lower specific gas productionrate, since the methane-forming bacteria have slow growthrates and need more time to grow-up.

Previous studies have reported that Methanosarcina spp.were always found to be the main acetate utilizer in biogasplants operating with sludge or manure, accompanied by lowlevels of acetate [4, 10]. It was also reported that the inoculumpopulation had almost no influence on the eventual popula-tion [10]. On the other hand, the characteristics of the inocu-lum might also influence the eventual population [24]. Sinceno microbiological investigation of our inoculum was carriedout, the effects (if any) of the inoculum on the eventual popu-lation within the biogas digester could not be clarified. Beforethis investigation was conducted, the reactor was run andstabilized for more than 4 years with silaged beets as a mono-input [15]. Furthermore, except for the failure situation onday 15 of HRT operation, the biogas digester was always oper-



ated with low VFA concentrations, but in an OLR range be-tween 3 and 4 g VS/L d.

In a recent study, the microbial community analysis of astirred tank reactor, fed with fodder beet silage as the sole sub-strate, was investigated for biogas production [13]. Using geneprobes, the authors qualitatively reported the presence ofMethanobacteriales (H2/CO2/formate-oxidizing), Methanosar-cinaceae (H2/CO2 oxidizing), or Methanosaetaceae (acetate-splitting) during the start-up phase (in the presence of man-ure). However, since the fodder beet (with leaves) and thesugar beet (without top leaves) have different characteristics(the buffering capacity and the nutrient availability of sugarbeet is poor-deprived), and a completely manure eliminatedbiogas reactor was investigated in this present work, the resultsfrom both studies cannot be directly compared.

Actually, the bacteriocenosis of biogas reactors running onfodder beet silage as the sole substrate seemed to be morediverse than that of sugar beet silage digesters, due to thehigher amount of nutrient availability [16, 24]. Furthermore,automatic feeding of fodder beet silage fed biogas digestersusing a fuzzy logic technique provided a more stable microbialecology in the digesters as well [24].

4 Summary

The continuous fermentation of acidic, low buffered sugar beetsilage was successfully performed. During mesophilic anaero-

bic digestion of the sugar beet si-lage as the sole substrate (withoutany manure addition), the highestspec. GPR of 0.72 L/g VS d wasachieved at an HRT of 25 days anda pH of around 6.80, with a biogascontaining 63 % of methane, usingsubstrate charge-2, which had anatural increase in its phosphatecontent. The digester was operatedat an OLR of 3.97 g VS/L d, appliedonce a day, and the volumetric gasproduction rate was 2.86 L/L d. Ahigher methane content in the bio-gas digester (67 %) and volumetricgas production rate (3.44 L/L d)obtained on day 15 of the HRT wasdue to higher loadings. The changeof the substrate charge from 1 to 2seemed to affect the methanogenicpopulation dynamics positively,thus, improving the reactor perfor-mance significantly. Besides, achange in the HRT did not seem toaffect the morphology of themethanogenic population signifi-cantly. However, a decrease in theHRT seemed to affect the cell num-ber of the slow-growing methano-genic population adversely. Sincesugar beet silage (without top and

leaves) is a relatively poor substrate, an external supply of buf-fering agents and nutrient sources is required for a stable andsafe digestion process for the continuous production of biogas.Recirculation of the reactor content and an automatic systemwith an increased feeding frequency per day, on the otherhand, could stabilize the anaerobic digester [15, 27, 28]. Theuse of a new charge of substrate (a new harvest) with a higherphosphate content improved the performance of the biogas di-gester significantly.

Acknowledgements

The authors would like to express their gratitude to NilsSharfenberg and Christian Rösner for chemical analyses. Theauthors would also like to thank Karsten Lehmann and OlafSchmidt for their technical assistance during reactor manage-ment, and Monika Unbehauen, for the general technical sup-port. This project was supported by the BMBF KFZ 03SF03171.

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Figure 6. The changes in the numbers of the total cell count and the fluorescent methanogens(n = 3) at 25 and 15 days of the HRT with the same substrate charge-2 under steady-state condi-tions.


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