Dry Anaerobic Digestion of Cow Dung for Methane Production: Effect of Mixing
Ajay Kumar Jha,
The performance characteristics of a dry batch reactor with a blender treating cow dung has been evaluated for 35 days in a single-stage batch reactor of 3 L effective volume at 35±1°C to investigate the effect of continuous-mixing on biogas production and organic materials removal. The results showed that the performance of unmixed and mixed digesters was quite different and the dry digester with mixing system produced methane of 0.358 LCH4/gVSr which was 7.50% higher than that for unmixed digester. Moreover, the organic material removal efficiency was increased by 9.73% in term of VS. The wide diversity of prominent bacteria and methanogenic archaea affiliated with all steps along the anaerobic degradation pathway made the process stable. But the dry digester with mixing system during start up was not beneficial, as it resulted in relatively higher volatile fatty acids, higher volatile fatty acid to alkalinity ratio, lower pH and consequently prolonged start up time.
Received: December 01, 2012;
Accepted: February 06, 2013;
Published: March 09, 2013
Dry anaerobic digestion is an alternative to conventional manure management,
alleviating health and environmental concerns and converting organic wastes
(TS>10%) by microbial consortia in oxygen-free environment into biogas (Jha
et al., 2011). Besides reactor type, retention time, loading rate,
quality of feedstocks, environmental conditions within the digester and other
related parameters, the performance of an anaerobic digester depends upon the
degree of contact between the substrate and a viable bacterial population (Karim
et al., 2005; Kalia and Singh, 2001).
Adequate mixing can enhance biogas production and biodegradability (Kalia
and Singh, 2001) due to the distribution of substrates, enzymes and micro-organisms
throughout the digester. Mixing also promotes heat transfer and particle size
reduction, discharges gas bubbles trapped in the medium and avoids the sedimentation
of denser particulate matter (Kaparaju et al., 2008;
Ward et al., 2008). Although many researches (Karim
et al., 2005; Kaparaju et al., 2008;
Kalia and Singh, 2001) have reported about the importance
of mixing in achieving efficient substrate conversion in wet anaerobic digesters,
there is not a clear picture about the consequence of mixing on dry anaerobic
digestion of manure. In the present study, the effect of continuous mixing on
dry anaerobic digestion of cow dung for biogas production and organic materials
removal has been investigated.
MATERIALS AND METHODS
Experimental set up and procedure: The experiment was conducted into a single-stage batch reactor with mixing system at 35±1°C. The capacity of the reactor was 3.6 L with 3 L effective volume (Fig. 1). The produced biogas was escaped through a pipe into a water lock (3.0 pH) and then into a wet gas meter and finally released into the atmosphere. The samples for detecting various parameters were taken out from the side-ports. The samples were stored at -4°C in a freezer before analysis. In the fermentation process, the substrate was fed into the airtight digester under specified environmental conditions for 35 days without dilution. The digester was purged with nitrogen for 15-20 min to create complete anaerobic environment.
Characteristics of feedstocks: The cow dung was obtained from a livestock
farm of Harbin, China and made free from foreign materials including stone,
wood, metal, straw, feather and other inorganic materials, manually. The cow
dung was then inoculated with the 20% mesophilic digestate obtained from the
previous investigation of dry anaerobic digestion of cow dung.
|| Schematic diagram of the batch reactor with mixing system
The cow manure contained 16.28% total solids (TS),130.21 g Volatile Solids
(VS), 145.75 g Chemical Oxygen Demand (COD), 59.83 g soluble COD, 2.87 g L-1
nitrogen, 1.41 g L-1 phosphorus, 1.47 g L-1 ammonia nitrogen
and 0.85 g L-1 free ammonia per kg of the wet-manure. The high proportion
of VS to TS (84.3%) depicts that a large fraction of the manure was biodegradable
and could serve as an important feedstock for biogas production. The C: N of
the manure was found adequate (25:1) because it is often suggested that the
C: N ratio in the substrate should be in between 20:1 to 30:1. The pH of the
manure was also found favourable for the anaerobic digestion. In this study,
3 kg cow manure and 0.60 kg inoculant were mixed and incubated into the air-tight
dry digester with mixing system.
Analytical methods: The physico-chemical parameters analyzed were temperature,
pH, TS, VS, COD, soluble COD, Volatile Fatty Acids (VFAs), nitrogen, phosphorus,
ammonia nitrogen and free ammonia. All the analytical determinations were performed
according to the standard methods (APHA, 1995). All the
tests were conducted in triplicate and mean values were reported. The pH was
measured with a digital pH meter (Model 526, Germany). Free ammonia was calculated
using the formula described in the previous study (Ostergaard,
1985). The yielded biogas was measured daily using wet gas meter (LML-1,
Changchun Co. Ltd). The constituents (CH4, CO2 and H2)
of the biogas were determined using Gas Chromatography (SC-7, Shandong Lunan
Instrument Factory). The samples taken from the batch culture was centrifuged
at 6,000 rpm for 15 min and then acidified with hydrochloric acid and filtered
through a 0.2 μm membrane for the analysis of VFAs and ethanol. The concentrations
of the VFAs and ethanol were determined using a second gas chromatograph (Model
GC122, Shanghai Analysis Instrument Factory).
Microbial community analysis: Genomic DNA of the sludge samples was
extracted using a DNA extraction Kit (MO Bio Laboratories, Inc., Carlsbad, CA,
USA) following the manufacturers instructions. Extracted DNA was dissolved
in 60 μL 1xTE buffer solution. The V3 and V4 regions of 16S rRNA were amplified
by PCR using universal bacterial primers (341F, 5'-CCTACGGGAGGCAGCAG-3' with
a GC clamp and 907R, 5'-CCGTCAATTCMTTTGAGTTT-3') and universal archaeal primers
(344F, 5'-ACGGGGYGCAGCAGGCGCGA-3' with a GC clamp and 915R, 5'-GTGCTCCCCCGCCAATTCCT-3').
The PCR amplification was conducted in a 50 μL system containing 5 μL
10 x Ex Taq buffer, 4 μL dNTP mixture (2.50 mM), 1 μL forward primer
(20 μM), 1 μL reverse primer (20 μM), 2.5 ng DNA template and
0.15 U Ex Taq DNA polymerase (Takara, Dalian, China), using a thermal cycler
(model 9700; ABI, Foster, CA, USA), started with an initial denaturation of
DNA for 10 min at 94°C, followed by 30 cycles for 1 min at 94°C, 30
sec at 55°C (decreasing by 0.10°C per cycle to 52°C) and 1 min at
72°C; final extension was 10 min at 72°C. The PCR products were separated
using the Dcode universal mutation detection system (Biorad Laboratories,
Hercules, CA, USA). Polyacrylamide gels with 40-60% vertical denaturing gradient
were prepared. The 10 μL PCR products were loaded and electrophoresed at
120 V and 60°C for 10 h. Gels were stained silver as described in the previous
research (Bassam et al., 1991). All DGGE bands
were excised and dissolved in 30 μL 1xTE at 40°C for 3 h and then centrifuged
at 12000 rpm for 3 min. The 3 μL supernatant was used as the template and
conducted PCR amplification under the conditions as above described using the
same primers. The PCR products were pured by Gel Extraction Mini Kit (Watson
biotechnologies. Inc, China) and ligated into pMD18-T vector (Takara, Dalian,
China) and then cloned into E. coli DH5α. Some white clones from
each sample were randomly selected for PCR detection and positive clones were
selected for sequencing by ABI3730 and partial 16s rRNA gene sequences were
analyzed using the BLAST program in GenBank at National Center for Biotechnology
RESULTS AND DISCUSSION
Process characteristics: Figure 2 presents evolution
of pH, NH3-N, free ammonia and VFAs. The pH of cow dung was initially
around 7.64. Though pH and temperature are homogeneously mixed inside the digester
with mixing system, the pH variation pattern was observed identical to the unmixed
system. That means the pH was decreased swiftly during start up phase of the
experiment due to the increase in VFAs production by acidogenic bacteria as
well as carbonic acid associated with the high concentrations of carbon dioxide
gas. The easily digestible fraction of organic matter was hydrolyzed and converted
to fatty acids rapidly. The decrease in pH value was observed more in the reactor
with mixing system than that in the unmixed reactor. The main reason for the
lower pH during the mixing was attributed to relatively high production of VFAs
as well as release of the H+ ions during ammonia stripping. This
result is consistent with the previous research (Kaparaju
et al., 2008) which stated that vigorous mixing disrupts the structure
of microbial flocks and affect digestion efficiency. The pH was began to rise
gradually as the VFAs were consumed by methanogens and transferred to the methane.
It was also observed that there was stable pH after 4th week. The substrate
was able to buffer itself and prevent the acidification occurrence during digestion
due to proper alkalinity of cow dung, which is a pre-requisite for proper biogas
production. The phosphorus, nitrogen and ammonia nitrogen were noted 1.41 to
1.13, 2.87 to 1.79 and 1.52 to 1.43 g L-1, which are sufficient to
satisfy the cell growth requirements for biogas production. Variation in ammonium
nitrogen levels was relatively lower compared to unmixed system because of stripping
of ammonia in continuous mixing of the substrate inside the digester. The ammonia
concentrations were noted below the inhibitory levels as the critical ammonia
concentration is 2.8 g L-1 (Poggi-Varaldo et
||Evolution of (a): pH, (b): Ammonia nitrogen and free ammonia,
(c): VFAs and (d): VFAs/alkalinity in the reactor with mixing system
||(a): Daily biogas production and its methane content in the
reactor with mixing system and (b): Comparison of cumulative biogas yields
in the reactors with or without mixing system
Free ammonia is the active component causing ammonia inhibition (Hansen
et al., 1998). The calculated free ammonia ranged from 0.01 to 0.08
g L-1 in the reactor, indicating no possibility of inhibition in
the process due to existence of free ammonia in the reactor with blending system.
The value obtained was not supposed to be high enough to create inhibition as
though ammonia can inhibit fermentation process; the total ammonia concentration
that can be tolerated was relatively high. Volatile fatty acids are usually
produced due to the degradation of the complex organic polymers during hydrolysis
and acidogenic stages. The conversion of intermediate products-VFAs-has been
treated as an indicator of the digestion efficiency but the high concentration
of VFAs results in decrease of pH, inhibit acidification, destroy methanogenic
bacteria activity and leading to failure of digester ultimately. In this study,
the reactor showed high volatile fatty acids concentrations in the start up
phase (Fig. 2c) due to higher acidogenesis and lower methanogenic
activities. The principal volatile fatty acids formed were acetic, butyric and
propionic acids. Acetic acid was the dominant volatile fatty acid. The share
of propionic and butyric acids was observed low because of the sufficient propionate-
and butyric-degrading syntrophs which could rapidly convert propionic acid and
butyric acid to acetic acid (Montero et al., 2008).
The VFAs were increased rapidly after starting the test and reached a maximum
of 10.33 g L-1 after 7 days. During this period, the acetic acid
production rate was apparently higher than the acetic acid consumption rate.
The degradation of propionate and butyrate by syntrophic acetogenic bacteria
produced acetic acid that was subsequently degraded into methane and CO2
by acetoclastic methanogens (Montero et al., 2008).
During methanogenic stage, acetic acid was started to convert into biogas such
as methane and carbon dioxide. Thus, as methanogenesis and methane gas yield
have increased, the VFAs concentrations were decreased. No high VFAs accumulation
was detected due to perhaps acetatoclastic methanogens could consume acetate
quickly in the digester to yield methane and carbon dioxide. At the end of the
process, VFAs concentration was decreased to 2.18 g L-1. The ratio
of VFAs to alkalinity was in favorable range during the digestion period but
it was more than 0.80 on the 7th day. It means the reactor was slightly inhibited
during the first week (Sanders et al., 1996).
Afterwards, the process seemed stable as no accumulation of VFAs and fall in
pH were observed and consequently no significant inhibition during the digestion
Enhancement of biogas production and biodegradability: Figure
3 shows the daily biogas production and its methane content in the reactor
with mixing system. The biogas generation was started after seeding, kept increasing
until reaching the peak and then began to decline. The biogas production and
methane yield patterns were found similar to the previous unmixed mode (Jha
et al., 2010; Li et al., 2011). It
means the biogas and methane generation showed an increase day after day until
reached the maximum value and then decreased slowly day after day but biogas
production was detected low in the reactor with mixing system during first week
of the fermentation process. It happened due to high VFAs production, decrease
in pH value and lack of methanogens. This result indicated that the reactor
with the mixing system for treating cow dung could not be favourable during
the start up period.
|| Comparison of characteristics of mixing system with unmixed
|| Total solid and removal of VS in the reactor with mixing
system during the fermentation period
The cumulative biogas generation was measured 47.56 with 26.72 L kg-1
methane content during 35 days of the digestion period. Comparing with the previous
unmixed bioreactor (Table 1), additional 7.50% biogas was
noted in the reactor with mixing system. The percentage of extra biogas was
not so high because the unmixed reactor was also actually mixed 2-3 minutes
daily. No significant change was observed in the quality of biogas.
The initial methane contents in the yielded biogas has increased and exceeded
to the peak value and then decreased slowly to some extent. The hydrogen content
in the biogas was observed negligible as like the unmixed bioreactor. Figure
4 also presents VS removal efficiency in the reactor with mixing system
during the digestion period. The VS was degraded about 41.90% in the reactor
with mixing system. It was degraded about 38.19% in the previous study of dry
fermentation process of cow dung in the reactor without mixing system for the
same digestion period (Li et al., 2011). Thus,
the mixing strategy could boost VS removal efficiency by 9.73%. The VS removal
has close relation with the methane yield. Therefore, the percentage of VS removal
was increased as cumulative methane production was increased.
Microbial community analysis: The sludge was sampled for PCR-DGGE analysis
on the seventh day and at the end of fermentation process (digestate) to understand
the potential linkage between the bacterial and archaeal community structure
and digestion process performance. The thirteen prominent bands were obtained
from DGGE for bacteria (Fig. 5a) and then sequenced. The phylogenetic
analysis of the representative bacterial clones revealed that micro-organisms
in the phyla Firmicutes, Bacteroides, Proteobacteria and Ruminobacillus were
observed (Table 2). Among them, the phylum Firmicutes was
dominant group and within phylum Firmicutes, class Clostridia was the
most dominant of the bacterial community. Micro-organisms within the class Clostridia
and Bacteroidetes have been frequently reported to be important throughout
various anaerobic habitats and have the ability to degrade a wide variety of
complex organic molecules, including proteins and carbohydrates. Clostridum
and Bacteroides species isolated from rumen, digesters and natural habitats
hydrolyze cellulose, hemi-cellulose and protein to produce VFAs, alcohol, CO2
and H2. Most of the members of the genus Clostridium are strictly
anaerobic, producing ammonia, H2S and H2 and ferment carbohydrates
into acetate, butyrate, ethanol, CO2 and H2. Therefore,
the higher VS reduction, obtained might be due to the high abundance of these
bacteria. In this study, the identified micro-organisms within these classes
are in agreement with other community analyses in anaerobic digesters and demonstrate
the importance of these phylogenetic groups, for the degradation of complex
organic matter in the fermentation process systems. There were eleven prominent
bands obtained from DGGE in archaea domain (Fig. 5b) and PCR-DGGE
patterns of different DNA fingerprint bands represented the different microbial
species of the 16S rRNA. DGGE profile clearly reflected the shift in microbial
communities and the number of bands observed at the end of experiment for archaea
was more than those for the initial stage (on the 7th day). Euryarchaeota was
the most abundance archaeal 16S rRNA gene sequences and classified into Methanomicrobiales
and Methanosarcinales. Hydrogenotrophic methanogens, such as Methanoculleus,
Methanogenium and Methanobrevibacter, dominated the archaeal
communities along with some aceticlastic methanogens.
||DGGE fingerprints of the samples (a) Bacteria and (b) Archaea
in the reactor with mixing system, (S1): Sample of the 7th day, (S2): Digestate
|| Bacterial 16S rRNA gene clone libraries in the dry methane
fermenter with mixing system, compared by BLAST with NCBI
Methanoculleus bourgensis, Methanosarcina barkeri, Methanospirillum
hungatei and Methanomicrobiales archaeon were the most abundant methanogenic
species in the fermenter (Table 3). Each of them showed some
specific characteristics in methanogenic metabolism. Methanoculleus bourgensis
was reported to use H2-CO2 or formate as a substrate for
growth and methanogenesis and is a hydrogenotrophic methanogen. Methanospirillum
hungatei produces methane only from H2-CO2 or formate,
but not from acetate or ethanol and methanol, being a strictly hydrogenotrophic
methanogen. Methanosarcina barkeri could be used in different substrates
to produce methane, including H2-CO2, methanol, mono-,
di- and trimethylamines, acetate and CO and is a hydrogenotrophic or aceticlastic
|| Archaeal 16S rRNA gene clone libraries in the dry methane
fermenter with mixing system, compared by BLAST with NCBI
The prominent bacteria and methanogenic archaea that can be affiliated with
all steps along the anaerobic degradation pathway of organic matter to methane
have been detected. The high diversity and dynamic activity of methanogens is
favorable for maintaining the efficiency of the fermentation process.
Mixing appeared to be necessary for effective operation of biogas plants and
consistency of feedstock fermentation inside the reactor because it created
close connection between micro-organisms and substrate, even distribution of
pH and temperature. High diversity of bacteria and archaea supported favorable
anaerobic condition within the reactor. Though the performance of the dry methane
fermenter with mixing system has poor in the first week due to higher production
of VFAs and consequently lower pH, continuous mixing strategy had provided 7.50%
additional methane yield and 9.73% higher volatile solid removal efficiency
during the fermentation period of 35 days compared to the unmixed dry methane
fermenter. In order to prevent higher accumulation of VFAs during start-up phase,
intermittent-mixing may be useful.
The authors would like to thank the State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (Grant No. 2010DX06), National S&T Major Projects (Grant No. 2008ZX07207-005-02) and Harbin Science and Technology Bureau (Grant No. 2009RFXXS004) for their support for this study. In addition, International Student Center is specially thanked for providing financial support to take part into the conference.
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