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Efcacies of inocula on the startup of anaerobic reactors treating dairy manure under stirred and unstirred conditions
Pramod K. Pandey a,*, Pius M. Ndegwa b, Michelle L. Soupir a, J. Richard Alldredge c, Marvin J. Pitts d
a

Department of Agricultural & Biosystems Engineering, Iowa State University, Ames, IA, 50011, USA Department of Biological Systems Engineering, Washington State University, Pullman WA, 99164 -6120, USA c Department Statistics,Washington State University, Pullman, WA 99164-3144, USA d School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164-2920, USA
b

article info
Article history: Received 24 December 2009 Received in revised form 19 February 2011 Accepted 4 March 2011 Available online 6 May 2011 Keywords: Inocula Anaerobic digestion Dairy manure Mixing Syntrophobacter Methanogens

abstract
Inocula play an important role in anaerobic reactor startup by balancing the populations of Syntrophobacter and methanogens. Such balances make syntrophic metabolism thermodynamically feasible in anaerobic digestion. In this study, the effect of inocula on the performance of dairy manure digestion was investigated by analyzing the change in volatile fatty acids (VFA), total solids (TS), volatile solids (VS), specic biogas production (SGPR), and specic methane production (SPMP) as well as scanning and transmission electron micrographs. The study was performed at four treatments. Treatment one was granular sludge (GM); treatment two was non-granular sludge (SM); treatment three was mixed culture from an anaerobic lagoon (LM); while the fourth treatment (the control denoted MM) did not receive any exogenous inocula. In addition, stirred and unstirred conditions were maintained in the reactors to determine their effect on reactor startup. Performance ranking based on the SGPR and SPMP of treatments (in descending order) was: GM, SM, LM and MM under stirred conditions. Under unstirred conditions, performance ranking (also in descending order) was: SM, GM, LM, and MM. Results of the examination of microcolonies in the granular, non-granular sludge, and dairy manure suggest that syntrophic juxtaposition of methanogens and Syntrophobacter in granular inoculum was common while it was less visible in non-granular sludge, and completely absent in dairy manure. Published by Elsevier Ltd.

1.

Introduction

The United States is the largest energy consumer in the world [1] and obtains most of its energy from non-renewable fossil fuels. In principle, the country could signicantly reduce consumption of non-renewable energy by using waste products such as dairy manure as a source of energy. Dairy manure is a renewable energy source in many less-developed countries, where economic conditions limit the availability of fossil fuel. This utilization of animal waste as an energy

source has been realized via anaerobic digestion that converts animal waste into biogas, which is subsequently used as fuel for heating and cooking. Besides generating biogas for energy use, the anaerobic process also reduces pathogens and produces stabilized slurry to be used as fertilizer in land applications [2]. In the U.S., dairy cattle generate an estimated 2 1012 kg of solid waste annually [3] and could provide a signicant source of renewable energy in the form of methane. If this volume of dairy waste can be used for biogas production, it has a potential to generate around 8 1010 m3 of

* Corresponding author. Tel.: 1 515 894 2210; fax: 1 515 894 4250. E-mail address: pkpandey@iastate.edu (P.K. Pandey). 0961-9534/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.biombioe.2011.03.017

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Nomenclature GM SM LM MM F/M a VFA TS VS SEM treatment in which manure and granule was mixed treatment in which manure and non e granular sludge was mixed treatment in which manure and anaerobic lagoon culture was mixed treatment which did not receive any exogenous inoculum food to microorganisms ratio ratio between VFA and bicarbonate alkalinity volatile fatty acids total solids volatile solids scanning electron micrographs

TEM SPMP SGPR TAN VSfeed VSinocula COD Msae Mbac Dsv Dsb Msar Msp Synw

transmission electron microscope specic methane production specic gas production rate total ammonia nitrogen volatile solids in feed (manure) volatile solids in inocula chemical oxygen demand Methanosaeta spp. Methanobacterium spp. Dsulfovibrio spp. Desulfobulbus spp. Methanosarcina spp. Methanospirillum spp. Syntrophobacter wolinii

biogas annually (annual solid waste: 2 1012 kg; biogas yield: 0.040 m3/kg) with an energy value of 1.6 1012 MJ (caloric value of biogas: 20 MJ/m3) [4]. Given that one person in the U.S. uses about 361303 MJ primary energy [1] annually, biogas produced from dairy waste can thus fulll the annual energy need of 4.42 million people. However, so far manure is largely underutilized as a fuel source because of the problem and complexity involved in the anaerobic digestion process required for gas production. Anaerobic digestion is a conversion of biodegradable material that involves hydrolysis, acidogenesis, acetogenesis, and methanogenesis processes. Hydrolysis/acidogenesis process involves the conversion of biodegradable material into volatile fatty acids (VFA) such as acetate, butyrate and propionate [5]. During the acetogenesis process, VFA is converted into acetate, hydrogen, and carbon dioxide. Methanogenesis process involves the conversion of acetate into methane and carbon dioxide. Table 1 shows the syntrophic and methanogens population of granule, manure and sludge. Compared to granule and sludge samples, manure samples constitute a higher number of syntrophic bacteria such as propionate and butyrate degraders. The aceticlastic methanogens are reported to be higher in sludge samples than granule and manure samples, and hydrogenotrophic methanogens are reported to

be less in sludge samples than granule and manure samples. While, acetogenesis and methanogenesis can be considered as the major rate-limiting steps during anaerobic digestion of complex waste (cattle manure), the VFA produced by hydrolysis/acidogenesis can depress the acetogenesis and methanogenesis [5,6]. To balance the VFA production and consumption, a balance population of these two groups of microorganisms (acetogens and methanogens) is required. If hydrogenotrophic methanogens fail to consume the H2 produced during acetogenesis, accumulation of H2 concentrations occurs, which results in increased H2 partial pressure. The increased H2 partial pressure limits the anaerobic biodegradation of VFA [2,7,8,9,10]. To start gas production in reactors (reactor startup), a balanced population ratio of acetogens and methanogens is required. The requirements of a balanced population and the slow growth rate of methanogens are the major problems in reactor startup. The initial 1e3 weeks are considered as the reactor startup period, in which biomass acclimatizes and grows in a new environment [11,12,13]. The slow growth rate of methanogens extends the time required for establishing a dynamic equilibrium between acetogens and methanogens, which results in an accumulation of intermediate degradation products (VFAs and dissolved H2) [14]. Excess VFAs inhibit the growth rate of methanogens by

Table 1 e Microbial community population in inocula. Bacterial groups

Syntrophic (A) *Propionate degraders Butyrate degraders Methanogens (B) **Aceticlastic methanogens Hydrogenotrophic methanogens

Granulara MPN/mL granules

3 108 1 108 3 109 3 109

Manure % SSU rRNA

1.03b 0.49b 0.21c 1.0c

Sludge % SSU rRNA

1.04b 0.42b 9.59c 0.19c

Granule % of (A B)
4.68 1.52 46.88 46.88

Manure % of (A B)
37.73 17.95 7.69 36.63

Sludge % of (A B)
9.25 3.74 85.32 1.69

*Sum of Syntrophobacter fumaroxidans, Syntrophobacter pfennigii and Syntrophobacter wolinii. **Sum of Methanosarcina spp. and Methanosaetaceae a Granule grown on starch industry waste water in mesophilic conditions. Maximum Possible Numbers (MPN) was used due to unavailability of 16S rRNA probes data on granule sludge [51,10]. b Microbial analysis of inoculum, expressed as percentage of specic SSU rRNA of total SSU rRNA (% SSU rRNA) [26]. c Methanogenic population levels in inocula, expressed as percentage of specic SSU rRNA of total SSU rRNA (% SSU rRNA) [8].

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lowering pH [15], which results in delayed startup or failure of the digester. Therefore, during the startup, the microbial community should contain sufcient levels of methanogens to avoid these problems. Poor startup in anaerobic treatment systems can lead to prolonged period of acclimation [16] and ineffective removal of organic matter [8]. Several studies reported that successful startup was related to a number of factors e.g., seed sludge source, initial loading rate, hydraulic retention time (HRT) or solids retention time (SRT) [8]. Extensive research has been done on the effect of initial loading rate, HRT, and SRT on anaerobic digestion. However, relatively little research has been reported on the effect of different inocula on startup of anaerobic digester treating dairy manure. The duration of startup phase depends on the type of inocula and feed materials. Several studies emphasize the importance of inocula in anaerobic digestion [17,18]. The use of anaerobic digesters sludge treating municipal waste water as an inocula is found to be suitable for the startup of the mesophilic anaerobic digester [19,8]. The use of granular sludge as inocula has been found to be suitable for treating pharmaceutical waste water [20]. However, the use of granular sludge in dairy manure treatment has not been reported. Despite a number of available studies suggesting the use of inocula for startup of the reactors [21], an exact specication or a study which mainly focused on selection of inocula for anaerobic digestion of treating dairy manure is lacking. The objective of this study was to investigate reactor startup using different inocula as well the effect of stirring during startup phase.

2.2.

Inocula

2.
2.1.

Materials and methods

Dairy manure (feed stock) characteristics

Raw dairy manure (pH 6e7) was collected from the Knott Dairy Centre at Washington State University in Pullman, WA and then stored at 20  C. Just before the treatments preparation, the samples were transferred from 20  C to 4  C. The feed was prepared by diluting the raw manure with water to desired feed concentrations (TS 2.5%, g/g). The feed was then sieved with a mesh of 850 mm (USA standard test sieve, No 20, S/N 04106855, Fisher Scientic Company) to remove large debris and ber in order to minimize blockage of the lab-scale reactors. The characteristics of dairy manure used in this study are presented in Table 2. Determinations of chemical oxygen demand (COD), TS, VS, and TAN were performed according to Standard Methods [22].

Three types of inocula were compared in this study. One; granular seeds, which were collected from a UASB reactor treating potato starch waste at Penford Food Ingredients Co, Richland, WA. Prior to being fed to the UASB reactor, the stream of potato starch waste was taken through a settling tank to allow solid particles to settle. Subsequently, liquid portion with a BOD of 4000 mg/L was fed to UASB reactors at a rate of 220 gallon/day. The UASB reactor with 4 h of hydraulic retention time produced about 1 million cubic feet of biogas per month and disposed the efuent with BOD of 100 mg/L. Two: Non-granular sludge of anaerobic digester, which was collected from the Pullman Sewage Treatment Plant, Pullman, WA. This treatment plant serves a population of approximately 24,360 and uses activated sludge waste water treatment with chlorine disinfections. The solids handling process consists of dissolved air otation unit, gravity thickener for primary slide, sludge equalization tank, anaerobic digesters, and sludge holding tank. The average inow is about 3.5 million gallon per day with an average BOD of 154.56 mg/L, and the treatment facility efuent BOD is about 4 mg/L. Three: Mixed anaerobic culture seed, which was collected from an anaerobic dairy waste lagoon located at the Knott Dairy Center, Washington State University, Pullman, WA. The 5900 m2 anaerobic lagoon receives ushed manure from a dairy with a herd of approximately 250 cows (milking cows and heifers) and approximately 80 calves. The waste consisting of feces, urine, bedding materials, and milking parlor waste is scraped into a manure pit from where it is subsequently ushed and delivered to a solids separator using recycled waste water. After the solid separator has removed solids of greater than 0.3 cm diameter, the waste water is pumped to the lagoon. The supernatant water from this lagoon was used as inocula. All inocula were stored in 20  C and were transferred to 4  C just prior to treatments preparation. The inocula characteristics are shown in Table 2.

2.3.

Experimental design

The study was designed to investigate the efcacy of granular and non-granular sludge as well as the effect of mixing on the startup of anaerobic reactors. Inocula availability and possibility to use in full-scale reactors were the criteria for selecting the types of inocula for this study. Three different inocula and a control were investigated in mixed and non-mixed reactor startups. The experiment had the following four inocula treatments for the stirred and unstirred conditions mixed

Table 2 e Characteristic of inocula and dairy manure. Parameter

TS (g/g) VS (g/g) COD (mg/l) TAN (mg/l) Total VFA (mg/l)

Granular sludge
6.32 0.36 5.37 0.36 46393 2383 327 9 ND

Non-granular sludge
2.53 1.88 45303 887 302 0.19 0.08 1343 36 12

Mixed culture
1.62 1.30 21750 430 351 0.11 0.10 962 25 11

Diluted manure
2.25 1.68 30304 452 1130 0.17 0.09 1242 22 25

Raw manure
15.9 0.6 13.9 0.6 ND ND ND

Diluted manure was used for treatment preparations.

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with dairy manure into ratio of 1:4: (1) granular inoculum (GM), (2) sludge inoculum (SM), (3) anaerobic lagoon (LM), and (4) pure dairy manure (MM) with no exogenous inoculum (the control). In vitro anaerobic digestion tests were carried out in identical batch reactors having a working volume 125eml (Serum bottles, Scientic Instrument Services, Ringoes, NJ). The reactors were sealed with a thick rubber septum and then ushed with a gas mixture of 80% N2 and 20% CO2 for 5 min. Each treatment used 10 reactors resulting in 40 reactor units for stirring and 40 non-stirred reactor units. An orbital shaker (New Brunswick Scientic, Edison, NJ) at 125 rpm was used for stirring the reactors. The anaerobic digestion was carried out for ten days at 35  C. Digested slurry from one of the reactors (out of the 10 used per treatment) was used for digestion analysis every day; the reactor was subsequently discarded. The gas produced was measured with a gas tight glass syringee35 ml (Micro-Mate, interchangeable hypodermic syringe, Popper & sons, Inc. New Hyde Park, New York) and collected in a test viale10 ml (Labco Limited High Wycombe, 10 ml) every day. The gas was released after the volume measurement to avoid build-up of high pressure in the reactor that would lead into leakage of gas. This procedure was replicated in all the 4 treatments and 10 days of experimentation. Specic biogas production (SGPR, volume of biogas produced per ton of initial volatile solids per day), volatile fatty acids (VFA), pH, alkalinity, a (VFA to alkalinity ratio) [8,23], total solid (TS), volatile solid (VS), and total ammonia nitrogen (TAN) were measured in all treatment to evaluate the effect of inocula in reactor startup. The schematic of the experimental setup is presented in Fig. 1.

Varian FID for CH4, Varian TCD for CO2, and a De2 pulsed HID mode discharge detector for H2S measurement. The GC was equipped with a switching valve (A3C6UWT) with 1 mL sample loop and a column (Varian 180 180 Hayesep Q 80/100 Mesh Silcosteel and nitrogen as carrier gas) for CO2 and CH4 measurement. A switching valve (A3C6UWT) with 0.25 mL sample loop and a column (Varian 50 m 0.53 mm 4 mm SilicaPlot and helium as carrier gas) were used for H2S measurement. The oven and the TCD temperature were kept at 80 and 120  C, respectively. GC was calibrated with 99.9% pure CH4, CO2, H2S and H2 standards.

2.4.2.

Volatile fatty acids

The concentrations of volatile fatty acids (acetic, butyric and propionic acids) were determined using Dionex Ion chromatograph (DX-500 IC). The IC was equipped with AS-3500 autosampler. A small sample (feedstock, inoculum and digested slurry) was centrifuged at 12,000 rpm for 6 min. After centrifuging, supernatant was ltered through a 0.2emmeporeesize lter before injection into an IC. The Peak Net R software controlled the injection. A detector (ED 40 electrochemical detector) and a column (4 250 mm Carbon Pac PA 10 analytical column) were used for analyzing VFA concentrations. Elution was initiated with 10% (v/v) water and 90% (v/v) 52 mM NaOH for 27.10 min, with 100 ml of sample injected at 0.10 min. The elute ow rate was 1.2 ml/min with the pressure between 1500 and 3000 psi. The IC was calibrated with pure acetate, butyrate and propionate standards. A verication test was performed after every 10esample analyses.

2.4.3. 2.4. 2.4.1. Laboratory analyses Methane content

Scanning electron microscope analysis

Gas samples were withdrawn from the headspace of test vials and were injected into a Varian CPe3800 gas chromatograph (GC) for measuring the biogas contents (methane and carbon dioxide). The GC was equipped with following detectors:

Samples were rst xed overnight at 4  C with 2% paraformaldehyde, 2% glutaraldehyde and anaerobic 0.05 M cacodylate buffer for performing Scanning Electron Microscope (SEM). The xed samples were washed three times in an anaerobic 0.05 M cacodylate buffer and then, 1% osmium tetroxide (1e2%, for 1 h) was used for xing. The samples were then dehydrated with a graded series of ethanoledistilled water mixture (30e100% v/v) and placed in 100% ethanol. The samples were dried by the criticalepoint drying method before sputterecoating with gold particles. The samples were mounted on aluminum stubs and sputter coated in a Polaron E5100 (VG Microtech), with platinum/palladium target (60:40). The samples were then examined in a JEOL JSMe5800 LV scanning electron microscope.

2.4.4.

Transmission electron microscope analysis

Fig. 1 e Schematic diagram of experimental setup.

Samples prepared for transmission electron microscope (TEM) were washed with 0.1 M phosphate buffer (pH 7.2) and xed in 0.1 M phosphate buffer (pH 7.2) containing 2.5% glutaraldehyde for 12e16 h at 4  C. The xed samples were rinsed three times (10 min each) at ambient temperature in 0.1 mM phosphate buffer (pH 7.2) and postxed with 1% Osmium Tetroxide (OSO4) in the same buffer. The sample were then rinsed in 0.1 mM phosphate buffer, and dehydrated through a graded series (30e100%) of ethanol solutions, ethanoleacetone mixture (1:1, for 10 min) and through 100% acetone (two times, 10 min each). Dehydration was followed by sample ltration. A mixture of acetone and spurrs (1:1, for 1 h) and 100% spurrs (for 12e15 h) was used for ltration. Resin was changed prior

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to embedding. Samples were embedded in Polybed 812 (Polyscience, Inc., Warrington, Penn.). Thin sections were cut with ReicherteJung ultramicrotomes for resin sections and poststained with uranyl acetate and lead citrate. The samples were then examined in a JEOL 1200eEX, which was equipped with digital camera and X-ray microanalysis system.

9.1 (SAS Institute Inc., Cary, NC) was used for the analysis of the repeated measurements. Rank transformation approach was used to transform the data to a form more closely resembling a normal distribution framework [24]. Signicance was dened by p < 0.05. The developed statistical model for analyzing daily biogas production is presented in Eq. (1). Yijku mai bj abij ew gk agik bgjk abgijk es iju kiju (1)

2.5.

Statistical analyses

A mixed linear model was developed to examine and compare responses (biogas production) over time. The three factors examined in a completely randomized design (CRD) model were: (1) incubation period, (2) inoculum type (treatments), and (3) stirring conditions. The PROC MIXED function of SAS

Where: Yijku is biogas production in uth reactor (u 1,.,10) at ith treatment level (i 1,., 4); jth stirring level ( j 1(stirred),2 (unstirred); and on the kth day (k 1,.,10). The m is overall mean biogas production; ai is deviation of the response at ith treatment level from m; bj is deviation of the response at jth stirring level from m; gk is deviation of the response on the kth

Stirred reactors
350 300 250

Unstirred reactors
350 300

A
Biogas (ml/batch)
GM SM LM MM

B
GM SM LM MM

250 200 150 100 50 0

Biogas (ml/batch)

200 150 100 50 0

10

10

Specific biogas production rate ( m 3 biogas/ t VS)

Specific biogas production rate ( m 3 biogas/ t VS)

8000

8000

C
GM SM LM MM

6000

6000

GM SM LM MM

4000

4000

2000

2000

10

10

140 120

140 GM SM LM MM 120

Fraction of CH4 (%)

80 60 40 20 0

Fraction of CH4 (%)

100

100 80 60 40 20 0

GM SM LM MM

10

10

Days of incubation

Days of incubation

Fig. 2 e Gas prole of stirred and unstirred reactors.

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The cumulative biogas production (Fig. 2A and B), specic biogas production (Fig. 2C and D), and fraction of CH4 in biogas (Fig. 2E and F) were used to evaluate inocula performances. The repeated measure analysis of biogas production among the treatments is presented in Table 3. Comparison of biogas production is made among the treatments under both stirred and unstirred conditions. Under stirring conditions, during rst two days of incubation period, biogas production in GM treatments was signicantly higher than in the other treatments, and both GM and SM treatments produced biogas signicantly higher than the LM and MM treatments. Under unstirred conditions, biogas productions in GM and LM were not signicantly different. However, SM treatments produced signicantly higher amount of biogas than the other treatments. Overall, mixing increased gas production signicantly among all treatments during the rst four days except in the LM treatment, which produced almost similar amount of biogas in both stirring and unstirred conditions on rst day. Subsequently, however, the biogas production in stirring condition was higher than the unstirred condition. In stirred reactors, the maximum specic biogas production (SGPR) ( p < 0.05) for GM, SM, LM, and MM treatments was obtained on Day 1, 4, 10 and 10, respectively. The highest methane content in biogas of GM (77 1.04), SM (71 0.87), LM (37 3.64), and MM (33 1.37%) treatments was on Day 1, 3, 8 and 8, respectively. In unstirred reactors, the maximum biogas yield for GM, SM, LM, and MM treatments was obtained on Day 8, 3, 6 and 10, respectively. The highest methane content in biogas of GM (67 1.46), SM (70 0.66), LM (32 3.03), and MM (32 0.94%) treatments was on Day 10, 5, 8 and 10, respectively. Compared to other treatments, the gas production was delayed until Day 4 in MM treatments. The effects of mixing on treatment performance are shown in Figs. 2, 3 and 4. The mixing in anaerobic digestion is considered to be important and its effects are discussed extensively [27]. The study on effect of mixing which deals with inoculation and startup period, however, is rare. The

biogas production (ml)

Day 6

Day 5

21.58 Aa 10.65 Ba 30.57 Ca 7.67 Da M Mixing. NM No mixing. Data are compared for individual day column. Means in columns with different capital letters are signicantly different at p < 0.05. Means in rows with different small letters are signicantly different at p < 0.05. GM MM LM SM 54.80 Aa 1.50 Ba 19.70 Ca 8.30 Da 1.00 Ab 0.40 AB a 9.70 Cb 0.00 Bb 32.22 Aa 12.61 Ba 35.11 Ca 5.44 Da 5.11 Ab 4.22 Ab 24.74 Bb 0.00 Cb 25.01 Aa 11.37 Ba 40.60 Ca 6.76 Da 8.77 Ab 9.50 Ab 32.64 Bb 0.00 Cb 32.24 Aa 14.27 Ba 47.61 Ca 8.07 Da 9.21 Ab 11.29 Bb 31.31 Cb 3.36 Db

Table 3 e Repeated measures analysis of daily gas production.

Day 4

Day 3

Day 2

Day 1

TRT

NM

NM

NM

NM

13.55 Ab 11.67 Aa 28.38 Ba 3.08 Cb

NM

18.48 Aa 10.59 Ba 32.71 Ca 6.39 Da

18.89 Aa 12.00 Ba 20.65 Ab 3.81 Cb

NM

21.87 Aa 8.87 Ba 32.36 Ca 8.08 DB a

3.1.

Inocula performances

Day 7

23.74 Aa 10.50 Bb 21.78 Ab 1.75 Cb

NM

Startup period (time required to start biogas production) in anaerobic process is considered to be very critical [8] for anaerobic digester. This study is focused on the startup period of anaerobic digestion. Since the initial active biomass (population of Syntrophobacter and methanogens) plays a key role in catalyzing the anaerobic process [7,9,25,26], the performance of inocula (which was used as an initial active biomass to start the anaerobic process) was evaluated.

Day 8

15.11 Aa 7.31 Ba 25.19 Ca 10.29 Da

30.88 Ab 7.66 Ba 7.75 Bb 2.32 Cb

NM

2.28 Aa 8.70 Ba 23.77 Ca 11.41 Aa

3.

Results and discussion

Day 9

7.99 Ab 7.50 AB a 11.89 AC b 1.51 Db

NM

day from m; (ab)ij is an interaction term between ith treatment level and jth stirring level; (ag)ik is an interaction term between ith treatment level and kth day; (bg)jk is an interaction term between jth stirring level and kth day; (abg)ijk is a threeeway interaction term from ith treatment level, jth stirring level, and kth day. The ew and es iju kiju are mutually independent random errors; ew wN0;sw2 and es wN0;ss2 . iju kiju

Day 10

9.37 Aa 13.02 BC a 13.22 CD a 20.93 DE a

13.92 Ab 9.01 Bb 13.29 Aa 3.96 Cb

NM

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effect of mixing varies at different anaerobic stages (e.g., startup and steady state). In addition, it is altered by many other factors such as type of waste, type of inocula and changing food e microorganisms ratio (F/M) [27], which was evident in this study, too. In stirred condition, both GM and SM treatments produced better biogas than other treatments. However, under unstirred condition the SM treatment performed better than GM treatment, which indicates that stirring effect may vary for different inocula. The low gas production in treatments (GM, LM, and MM) under unstirred condition could have been the result of inadequate mixing to encourage the distribution of enzymes and microorganisms throughout the reactor [26,27,28,29]. The better performance of SM treatment in both stirred and unstirred reactors could be the result of higher level of aceticlastic methanogens (Table 1) [8,26,29]. In contrast to the stirred treatment, granule

settling in the unstirred treatment might have lowered the rate of the process. Higher settling characteristics of granule [8,10] may have resulted in poor contact between feed and inoculum. However, in some aspects, settling of the granule may be considered an advantage because it retains active biomass in the reactor [30]. Therefore, for a continuous or semi e continuous reactor, granules could be the best inocula, where a need to retain the active microorganism in the reactors for a longer time is necessary. The volatile fatty acids (VFA) concentration in the GM, SM, LM, and MM treatments are shown in Figs. 3 and 4. Two different patterns of VFA concentrations were evident for GM and MM treatments under stirred and unstirred conditions. In the stirred GM treatment, the VFA accumulation was comparatively very low, and the gas production was higher (with higher methane content). Under unstirred conditions,

5000

5000

VFA, Acetate (mg/lit)

4000 3000 2000 1000 0 0 5000 2

Stirred Unstirred

GM
4000 3000 2000 1000 0

Stiired Unstirred

LM

4 Stirred Unstirred

10
5000

10

VFA, Acetate (mg/lit)

4000 3000 2000 1000 0 0 2

SM

4000 3000 2000 1000 0

Stirred Unstirred

MM

10

10

Days of incubation
VFA, Propionate (m g/lit)
5000 4000 3000 2000 1000 0 0 2 4 6 8 10 5000 Stirred Unstirred SM 4000 3000 2000 1000 0 0 2 4 6 8 Days of incubation 10 0 2 Stirred Unstirred 3000 2000 1000 0 0 2 GM 5000 4000

Days of incubation
Stirred Unstirred

LM

10

VFA, Propionate (m g/lit)

5000 4000 3000 2000 1000 0

Stirred Unstirred

MM

4 6 8 Days of incubation

10

Fig. 3 e Acetate and propionate concentrations in GM, SM, LM, and MM treatments.

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5000

5000 Stirred GM Unstirred 4000 3000 2000 1000 0 0 2 4 6 8 10 5000 Stirred Unstirred SM 4000 3000 2000 1000 0 0 2 4 6 8 10 0 0

Stirred Unstirred

VFA, Butyrate (mg/lit)

4000 3000 2000 1000 0

LM

10

VFA, Butyrate (mg/lit)

5000 4000 3000 2000 1000 0

Stirred Unstirred

MM

10

Days of incubation

Days of incubation

Fig. 4 e Butyrate concentrations in GM, SM, LM, and MM treatments.

the initial increase in VFA concentration continued until Day 5 in the GM treatment, and until Day 10 in the MM treatment. The initial increase in the VFA concentrations indicates activity of hydrolytic and fermentative bacteria [8]. Compared to the GM and MM treatments, the VFA concentration was relatively the same in the SM and LM treatment under both stirred and unstirred conditions. As shown in Fig. 3, acetate was the main accumulated fatty acid. Compared to stirred conditions, the acetate concentration was higher in the GM and MM unstirred conditions. The acetate formation in anaerobic process is a result of propionate and butyrate conversion into acetate through syntrophic association [31]. The syntrophic association between propionate and butyrate edegrading bacteria with methanogens have previously been reported [10]. Table 5 shows the reactions and the free energy 0 change (D G0 ) during syntrophic metabolism. Such association, however, requires the favorable thermodynamics [32] and balanced population of methanogens as well as syntrophobacter. The whole anaerobic process starts with the syntrophic fermentative bacteria, which degrades propionate and butyrate (reaction 1e2) into the acetate and H2. However, these reactions are endogenic and thermodynamically unfavorable, unless the product, H2, is kept at low concentrations [7,32,33]. At low H2 concentrations endogenic reactions become exogenic (a thermodynamically favorable) [32]. This low concentration of H2 in anaerobic process is maintained by the hydrogenotrophic methanogens (reaction 3). These methanogens use the H2 to reduce CO2 to CH4 [32]. Finally, acetate is converted into methane by an exogenic process (reaction 4). Table 1 shows the reported syntrophobacter and methanogens in granule, sludge and dairy manure. Out of the combined population of methanogens and syntrophobacter, granule consists of 93.76% methanogens (aceticlastic 46.88% and hydrogenotrophic 46.88%). The sludge mostly consists of

aceticlastic methanogens (85.32%). Manure mainly consists of propionate and butyrate degraders [8,26]. The low population of methanogens could have been the reason for higher acetate accumulation in the MM treatments. In the GM and SM treatments, the low level of syntrophobacter and higher level of methanogens may have caused the low VFA concentrations and higher biogas production. The higher population of syntrophobacter may have caused the higher level of VFA in both the LM and MM treatments, which may have been the reason for low biogas production (Figs. 2, 3, and 4). Table 4 shows VFA/alkalinity ratio, which is denoted by a. The a indicates the balance between production and consumption of VFA [8]. Values of a less than 1.0 indicate acceptable ratios while values higher than the 1.0 signify imbalance between VFA production and consumption in the reactor, which may upset the anaerobic process [23]. PoggiVaraldo and Oleszkiewicz [23] and Grifn [8] proposed that an increase in the a-values precedes a pH decrease, making it possible to predict an imbalance before the VFA concentrations or pH reveal the instability. The a values were calculated for GM, SM, LM, and MM treatments under stirred and unstirred conditions. In stirred conditions, the lowest a value (0.07 0.12) was found in the treatment GM. This treatment produced the highest amount of gas. The second lowest a value (0.10 0.10) was found in the SM treatments, which produced second highest amount of gas. In LM and MM treatments, a-values were 0.56 0.12 and 0.69 0.18, respectively. The treatment with the lowest avalue produced overall the highest amount of gas. Similar results were observed under unstirred conditions. The SM treatment produced the highest amount of gas and had the lowest a-value (0.11 0.10). The highest a-value was observed in the MM treatment, which also produced the lowest amount of gas. Our results suggest that a-values can be used as indicators of potential biogas production in both stirred and

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Table 4 e Performance of treatments under stirred and unstirred conditions. Characteristics of treatments over 10 days of incubation
Acetic acid (mg/lit) Propionate (mg/lit) Butyrate (mg/lit) Total VFA (mg/lit) Alkalinity (mg/lit) pH TS (%) VS (%) VS/TS TAN (mg/lit) VSfeed/VS inoculum, F/M) SPMP (ml CH4/g VS) Alpha (a) SGPR (m3/t VS day) a TS Change (%) a VS Change (%)

Stirred GM
1

Unstirred LM
3

SM

MM

GM

SM

LM3
978 251 655 198 285 36 1892 522 3793 84 6.8 0.4 2.27(0.20) 1.61(0.15) 0.71 519 41 1.29 217 110 0.50 0.13 812 355 23 28

MM4
1859 439 704 142 327 77 2890 04 3342 99 6.3 0.1 2.15(0.23) 1.57(0.18) 0.73 379 49 1 26 21 1.23 0.10 147 117 30 33

326 58 327 234 186 ND 651 478 3559 516 5.8 0.5 1.64 (0.3) 1.19 (0.25) 0.73 386 97 0.31 1677 932 0.07 0.12 2215 1198 13 15

356 87 292 114 166 56 703 157 3823 292 6.9 0.6 1.71(0.32) 1.22(0.25) 0.71 587 48 0.89 1466 412 0.11 0.10 1960 651 45 43

1036 290 785 212 302 45 2096 443 3786 15 6.9 0.4 2.25(0.19) 1.45(0.50) 0.64 510 42 1.29 225 93 0.56 0.12 959 353 19 20

1025 281 794 220 263 114 2083 517 3024 181 5.9 0.5 1.76(0.17) 1.29(0.14) 0.73 460 45 1 106 72 0.69 0.18 694 329 18 21

1532 761 676 116 365 94 2469 976 4635 484 6.5 0.5 1.42(0.18) 0.72(0.24) 0.51 661 106 0.31 597 541 0.62 0.18 1213 23 21 70

356 107 252 105 205 ND 600 233 3743 42 6.9 0.6 1.73(0.33) 1.21(0.28) 0.7 549 41 0.89 943 418 0.11 0.10 1318 95 28 61

Superscripts in treatments (GM, SM, LM and MM) are performance ranking based on the specic biogas production (SGPR) and specic methane production (SMP). Treatments were prepared using 25% inocula and 75% feed (manure). The results of 10 day incubation were used to calculate the average and standard deviation (SD). ND indicates not determined. Total VFAs equals the sum of acetate, propionate, and butyrate. a Positive sign indicates increase while negative sign indicates decrease in TS or VS.

unstirred conditions. The MM unstirred treatment, with avalue of 1.23 (>1.0; the threshold of stability) produced the lowest amount of gas. Grifn [8] reported use of NaHCO3 for avalues higher than 1.0 to prevent reactors instability. Sung [35] proposed that a-values of about 0.10 can be considered as an indication for stable operation of mesophilic anaerobic digester. Our results corroborate Sungs recommendations. In our study, a-values were either less than 0.10 or about 0.10 in the GM and SM treatments. These two treatments produced higher amount of gas than the other treatments. Under stirred conditions, the average pH value in the GM treatment was 5.8 0.5. The pH-values ranged from 4.71 to 6.68 in the GM treatment; from 5.5 to 7.70 in the SM treatment, from 6.44 to 7.43 in the LM treatment, and from 5.47 to 6.76 in the MM treatment. Generally, the pH-values were higher than 6.0 during most of the incubation period for all treatments except GM stirred. The GM reactor, however, performed the best. Under unstirred conditions, the pH value ranged from 5.58 to 7.08, 5.8e7.64, 5.94e7.37, and 6.27e6.54, in the GM, SM, LM, and MM treatments, respectively. In previous studies, Zhang et al. [36] reported pH-values ranging between 6.1 and 6.8 during anaerobic of dairy manure, while Sung [35] reported pH-values ranging between 7.2 and 7.75 in mesophilic dairy manure anaerobic digestion. In our studies, pH-values usually ranged between 6.0 and 7.5. In general, low pH-values coincided with high a-values in most of the treatments. These results are similar to Sungs [35] results, who compared pH and a-values in thermophilic and mesophilic anaerobic digestion. The average TS and VS contents in all the treatments as well their respective changes during the treatments under stirred and unstirred conditions are shown in Table 4. The reductions in TS and VS were calculated as a percentage of the difference between initial and nal concentrations against

the initial concentrations. Under stirred conditions, TS and VS decreased in all treatments except the LM treatments. The respective decrease in TS and VS were 13 and 15% in the GM treatment; 45 and 43% in the SM treatment; and 18 and 21% in the MM treatment. In the LM treatment, the TS and VS increased by19 and 20%, respectively. In previous studies, Sung [35] also reported increase in TS and VS when reactors were performing poorly. In our studies, the LM treatment performance was relatively poor. Dugba and Zhang [37] reported TS reductions ranging between 6 and 28% and VS reductions ranging between 8 and 30% for dairy waste mesophilic anerobic digestion. The Dugba and Zhang study evaluated the impacts of organic loading rates on TS and VS removal and indicated that TS and VS reduction varied with loading rates. Their study also emphasized that longer incubation period may be required for signicant TS and VS reduction in dairy waste digestion. Under unstirred conditions, the respective reductions in TS and VS were 21 and 71% in the GM treatment; 28 and 61% in the SM treatments; and 30 and 33% in the MM treatments. In contrast, the TS and VS in the LM treatment increased by 23 and 28%, respectively. In general, approximately 5% more decrease in VS was observed compared to the decrease in TS in most of the treatments except GM and SM unstirred treatments. In GM and SM unstirred treatments, VS decreases were 70 and 61%, while decreases in TS were only 21 and 28%, respectively. High microbial activity in these two treatments may have increased VS reduction as well as improved gas production. These treatments produced higher amounts of gas than other unstirred treatments. Dugba and Zhang [37] also noticed that improved performance resulted in greater VS reduction. The VS/TS ratios for stirred and unstirred conditions are shown in Table 4. In GM and SM stirred treatments, the VS/TS ratios were higher than in the unstirred treatments. Both the

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GM and the SM treatments produced more gas in stirred conditions than in the unstirred conditions. In the LM stirred treatment, the VS/TS ratio as well as biogas production were lower than in the unstirred conditions. In the MM treatment, biogas production was comparable in both stirred and unstirred conditions, and VS/TS ratio remained same in both conditions. Based on the performance of the treatments, a ranking of suitable inocula was developed for inocula selection. The performance criteria were amount of gas production, methane content, and the onset of the gas production. Table 4 shows the performance ranking of the treatments. The ranking (from higher to low) for stirred and unstirred is GM, SM, LM, and MM, and SM, GM, LM, and MM, respectively.

3.2.

Microscopic examination of inocula

Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) examinations were performed on inocula (granular sludge, non-granular sludge and dairy manure) to determine the distribution of microbial population. The morphology of methanogens and other anaerobic microorganism published elsewhere [38,39,40,41,42,43,44,45,46] were used to identify the specic microorganisms in inocula. Granular inocula were spherical in shape and dark black in color. The diverse syntrophic colonies consisting of methanogens bacteria were present in the granules. Fig. 5A illustrates the external surface of granular inocula, whereas Fig. 5B illustrates a section of the granule. Randomly distributed cavities and holes with openings of 5e15 mm in diameter with microbial lining of Methanobacterium-like and Methanothrix-like bacteria were common (Fig. 5C), which facilitate gas or substrate transport from outer surface to inner part or vise versa [10]. While Methanobacterium are autotrophic organisms that grow under high H2 - pressure and consume H2 and CO2, Methanothrix are aceticlastic organisms, that play an important role in granule formation and use acetate [8,9]. The granule surface was predominant with two types of rods: small plump rods (Fig. 5C,D) and bamboo shaped small and long lamentous rods (Fig. 5D). Plump rods resembled Methanobacterium-like (Mbac) bacteria and bamboo shaped rods (Fig. 5D,E) resembled Methanosaeta (Msae; also knows as Methanothrix) - like bacteria [9,31]. Sectioned granule structures show the honeycomb structures at the centers (Fig. 5F) of the granules. These honeycombed networks are believed to be dead cells of the Methanothrix [44], which are responsible for acetate consumption. In the center of the granule, diatom like structures (Fig. 5G,H) were also found. These could be the viable diatoms enmeshed in granules that survived the extreme anaerobic dark environment [47,48]. TEM micrographs show the juxtaposing microcolonies in the granule (Fig. 6G). The colonies consisted of Methanospirillum (Msp)-like, Methanobacterium (Mbac)-like, and Methanosaeta (Msae)-like microorganisms (Figs. 6G, 7AeB). In addition, the colonies of Desulfobulbus (Dsb)-like fat rods and Dsulfovibrio-like (Dsv) (resembling half-moons and rough spheres) were observed occasionally. Also, the sheath like structure resembling Methanospirillum-like bacteria [31], hydrogen e and formate e utilizing bacteria [49],were randomly distributed (Figs. 6G and 7B). Microbial distribution in non-granular inoculum was different than in the granules. In the sludge, the bundles of

rodlike structures (Fig. 5I) consisting of Methanosaeta-like bacteria were observed. The long laments, identical to Methanosaeta, were abundant, which is common in sludge [29,46]. Filaments fused together resembling Methanosaeta-like bacteria, forming long rope-like structures, were predominant (Fig. 6A and B). These rope-like structures resembled Methanosaeta bers [34]. The Methanosaeta bre serve as initial nuclei for granule formation in anaerobic migrating blanket reactor (AMBR) and Upow Anaerobic Sludge Blanket (UASB) [46]. Presence of Methanosaeta bre in sludge, however, has not been reported. More observations of the sludge collected from different sludge digesters is needed to conrm that Methanosaeta bre formation exists in non-granule forming reactors. TEM micrographs show the presence of Methanobrevibactor-like rods (Fig. 7C) in non-granular sludge. These bacteria were in juxtaposition with other anaerobes. Sulfate reducing bacteria such as Dsulfovibrio and Desulfobulbus-like bacteria were observed in juxtaposition with Methanobrevibactor-like rods (Fig. 7D). Presence of Dsulfovibrio and Methanothermus fervidus bacteria presence in sludge are reported elsewhere [43]. Usually, Dsulfovibrio spp. form syntrophic association with the H2 utilizing bacteria (Methanobrevibactor) to produce acetate [31]. In addition, the juxtaposition of propionate degraders such as Syntrophobacter wolinii (Synw)-like and Methanobrevibactor-like bacteria with occasional presence of Methanospirillum-like and Methanobacterium - like bacteria was noted. In dairy manure, the Syntrophobacter-like bacteria were abundant. Dairy manure had less methanogens compared to sludge and granule inocula. In addition to Syntrophobacter and methanogens, dairy manure exhibited other structures such as ciliates and cocci. The cocci rods (25e40 mm in diameter) along with other cells and brous material were predominant on the surface of dairy manure (Fig. 6D). Some diamondshaped structures resembling Ostracodinium obtusum ciliates [42] were deeply embedded in the brous material (Fig. 6E). Structure of Syntrophobacter-like and Methanobrevibactor-like bacteria were present in unattached form (Fig. 8A,B). Methanobrevibactor are the main methanogens in dairy manure [40], however, Methanosarcina-like bacteria along the cist with holes and cavity were also noted occasionally (Fig. 6F). Methanobrevibactor are considered to be hydrogen and carbon utilizing bacteria and play an important role in methane formation (reaction 4 of Table 5). There are some reported studies which describe the somatic association between rumens ciliates and methanogens [50]. TEM micrographs show the random distribution of Syntrophobacter and methanogens (Figs. 7F, 8A and 8B). Fig. 7F shows the presence of Syntrophobacter and small rods resembling the Methanobrevibacter-like bacteria. Unlike in granules and sludge inocula, microcolonies formation was completely absent or very rare in manure and Syntrophobacter-like were more abundant (8A and 8B). Low population of methanogens may have been the main reason for delayed biogas production in MM treatments. In addition, higher growth rate of Syntrophobacter than methanogens may have further imbalanced the populations [27], which is a reasonable explanation for observed low and delayed biogas production in MM treatments. In granule inoculum, the juxtaposition of Syntrophobacter-like (propionate degrader)

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Fig. 5 e SEM of the granular sludge (AeH) and non-granular sludge (I). (A) External and (B) internal texture. (C) Cavities and hole surrounded with small plump rods (Methanobacterium-like) and long rods (Methanosaeta-like) at surface. (D) Aggregation of small plump rods at internal surface (Methanobacterium-like). (F)Honey comb structure, which believed to be formed by Methanosaeta-like bacteria. (G & H) Structures resembling diatoms was in the center of granules. (I) Bundle of rodlike structures in non-granular sludge.

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Fig. 6 e SEM micrographs of non-granular sludge (AeC), dairy manure (DeF) and a TEM micrograph of the granule showing the rods and cells (G). (A) Shows the small and long rods embedded in the lm. (B) Rope, which resembling Methanosaeta e bre. (C) Formation of long chain by Methanosaeta-like bacteria. (D) Micrographs showing long rods, short plump rods, and small cocci in the surface of dairy manure. (E) Ciliate-like structure in dairy manure in deep penetration of straw cells. (F) Methanosarcina-like structure in dairy manure (G) TEM micrographs of low magnications illustrating the microcolonies of different bacterial cells in granule.

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Fig. 7 e TEM micrographs of granule (AeB), non-granular sludge (CeE) and low magnication of manure (F). (A) Shows the juxtaposition syntrophic association in granule. (B) Shows the syntrophic growth of Dsulfovibrio-like (Dsv) and Methanobacterium-like (Mbac) cells. (C) Methanobacterium-like (Mbac) cells. (D) Juxtaposition syntrophic association of Methanobacterium, Desulfobulbus and Dsulfovibrio-like in sludge. (E) Dispersed (not in microcolonies) growth of Methanobacterium, Desulfobulbus and methanosaeta-like bacteria. (F) Show the abundance of syntrophobacter-like bacteria.

Fig. 8 e TEM micrograph of high magnications. (A) Shows the Methanosaeta (Msae), Desulfobulbus (Dsb), and Methanobacterium-like (Mbac) structure. (B) Contrast to granule, unorganized growth of cells in manure.

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Table 5 e Syntrophic reactions. Step Reactions D G0(kJ/mol)a

76.1

Involve Anaerobes
Syntrophic or proton- reducing acetogenic bacteria such as Syntrophobacter fumaroxidans [32] Syntrosphospora bryantii, Syntrophomonas wolfei [32,33] Hydrogenotrophic methanogens [32,33] Methanosaetaceae, Methanosarcina [8,32,33]

Acetate production in syntrophic metabolisms 1 CH3CH2COO- 3H2O / CH3COO- HCO3- H 3H2 Propionate / acetate (endogonic) 2 CH3CH2CH2COO- 2H2O / 2 CH3COO- H 2H2 Butyrate / acetate (endogonic) H2 consumption 3 4H2 HCO3- H / CH4 3H2O H2 / Methane (exogonic) Acetate consumption 4 CH3COO- H2O / CH4 HCO3Acetate / Methane (exogonic) a Change of standard Gibbs free energy in syntrophic metabolisms [32].

48.6

135.6

31.0

with Methanobrevibactor-like bacteria, and Syntrophomonas-like (butyrate degrader) with Methanobrevibactor-like bacteria may have balanced the production and consumptions VFA and H2, which have resulted in higher methane production. Compared to granules, the sludge exhibited a smaller number of microcolony formations between Syntrophobacter and methanogens, however, the higher availability of methanogens such as Methanosaeta may have helped in lowering VFA level and producing higher methane. SEM and TEM images were instrumental for comparing the different microbial shapes in granular, non-granular inocula and manure samples, and provided information on bacteria juxtaposition and association. For example, SEM images of granules showed that the external surfaces had abundant loosely associated rod shape structures (Fig. 5C) around the cavities, while the inner structures were more enmeshed and condensed and bacteria strongly fused together. In digested non-granular sludge, the bacteria aggregated around solid or brous particles and a thin lm was observed (Fig. 5I), which signify that there could be biolm formation during digestion. In manure samples, bacteria were more dispersed, and juxtapositions are not as prominent as in granules and digested sludge. The TEM images, for example, Fig. 7A, showed that bacteria in granules were more closely associated and one group of bacteria was surrounded by the other (Fig. 7B). In digested sludge (Fig. 7D) bacteria were slightly farther apart but a thin lm, which could be biolm, surrounded the bacteria. In fresh undigested manures (Fig. 7E) bacteria were more independent and showed no indication of lm formation. Molecular based technology such as 16 sRNA is more appropriate for quantifying total number of bacteria. However, SEM and TEM images are more useful for elucidating the juxtaposition and syntrophic association among the various groups. These images were helpful in clarifying the population variation on the external and internal surface of granules and biolm formations. Several reports have been published, which explore the 16 sRNA techniques. Hwang et al. [52] used this technique to evaluate methanogenic population in anaerobic digestion of swine waste water; while Ike et al. [53] studied population dynamics during startup treating industrial food waste using the same technique. Others including Shin et al. [54,55] and Lee et al. [56] have also used this

technique: Shin et al. [55] performed comprehensive microbial analysis in food waste-recycling waste water, while Lee et al. [56] provided quantitative analysis of methanogens in batch digesters treating different waste water. These studies were important in understanding population dynamics. In view of this, future work conducted over varying digestion periods and digestion performance should combine SEM and TEM imaging and quantitative microbial analysis using 16 sRNA to enhance understanding microbial dynamics and interaction at optimal performance conditions. The idea of optimizing the ratio between syntrophic and methanogens population, which we propose in this study, would need further work. Future work should focus on providing precise quantication of these two groups of bacteria at optimal conditions. This information has the potential of enhancing anaerobic digester startup and subsequent operation.

4.

Conclusions

The efcacies of three different inocula in the anaerobic reactor startup treating dairy waste water were investigated under stirred and unstirred conditions. The results from this study suggest that selection of inocula is critical for anaerobic reactor startup. Overall, stirring enhanced the biogas production and lowered accumulation of the VFA. Stirring was more effective in GM and MM treatments than the SM and LM treatments. Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) analyses revealed abundant aceticlastic and hydrogenotrophic methanogens in granules, which may have provided ideal conditions for Syntrophobacter to efciently degrade propionate and butyrate without VFA accumulation. This could explain better biogas production and low VFA levels in treatments when granule was used as inoculum. Similarly, in SM treatments, a higher level of aceticlastic methanogens in the digested sludge, which consumes acetate, may have played crucial role in reducing the VFA accumulation and enhancing digestion performance. In MM treatments, low population of methanogens and higher population of Syntrophobacter may have lowered the biogas production and increased the level of VFA in the startup period. The SEM and TEM images showed the variation in juxtaposition and bacteria population level in different inocula, and

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these were linked with biogas production in corresponding treatments demonstrating the importance of syntrophic association and population level of different groups of bacteria in anaerobic digester performance. Future work should focus on providing better estimation of total population in each inocula using molecular based techniques such as 16 sRNA coupled with the SEM and TEM image analysis to enhance understanding of syntrophic interactions and role of each group of bacteria in anaerobic digestion.

[15]

[16]

[17]

Acknowledgments
This study was conducted with nancial support from Dr. Shulin Chens Research Program (Dr. Chen is a Professor of Biological System Engineering at Washington State University).

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