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Microbial pretreatment of water hyacinth for enhanced hydrolysis followed by biogas production
Accepted Manuscript Microbial Pretreatment of Water Hyacinth for Enhanced Hydrolysis followed by Biogas Production
Visva Bharati Barua, Vaibhav V. Goud, Ajay S. Kalamdhad PII:
S0960-1481(18)30334-3
DOI:
10.1016/j.renene.2018.03.028
Reference:
RENE 9897
To appear in:
Renewable Energy
Received Date:
07 October 2017
Revised Date:
04 January 2018
Accepted Date:
13 March 2018
Please cite this article as: Visva Bharati Barua, Vaibhav V. Goud, Ajay S. Kalamdhad, Microbial Pretreatment of Water Hyacinth for Enhanced Hydrolysis followed by Biogas Production, Renewable Energy (2018), doi: 10.1016/j.renene.2018.03.028
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2
Microbial Pretreatment of Water Hyacinth for Enhanced Hydrolysis followed by Biogas Production
3
Visva Bharati Baruaa,*, Vaibhav V. Goudb and Ajay S. Kalamdhadc
4 5
aCentre
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bDepartment
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cDepartment
1
for the Environment, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati781039, Assam, India of Civil Engineering, Indian Institute of Technology Guwahati, Guwahati781039, Assam, India
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*email: [email protected]
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ABSTRACT
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Biological pretreatment with novel isolated microbial pure culture was utilised to pretreat
13
water hyacinth to enhance its solubilisatio n followed by biogas production. Lignocellulose
14
degrading bacterial strains isolated from soil (Bordetella muralis VKVVG5) (UN3d2), the gut
15
of silverfish (Citrobacter werkmanii VKVVG4) (SFa2) and millipede (Paenibacillus sp.
16
VKVVG1) (BrB2) were employed to optimise the ideal bacterial strain illustrating accelerated
17
hydrolysis of water hyacinth. Citrobacter werkmanii VKVVG4 pretreatment of water hyacinth
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with an optimum dosage of 109 CFU/mL and time of 4 days helped in achieving the highest
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solubilisation of 33.3%. Biochemical methane potential (BMP) test was conducted between
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untreated and Citrobacter werkmanii VKVVG4 pretreated water hyacinth. Biochemical
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methane potential (BMP) test of pretreated water hyacinth illustrated faster start up period
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than the untreated water hyacinth. Citrobacter werkmanii VKVVG4 (SFa2) pretreated water
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hyacinth illustrated a cumulative biogas production of 3737±21 mL whereas untreated water
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hyacinth illustrated a cumulative biogas production of 3038±13 mL on the 50th day. Scaled
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up batch (20 L) study demonstrated a three times increase in the cumulative biogas
26
generation of the microbial pretreated water hyacinth than the untreated water hyacinth.
27
Keywords: Water hyacinth; lignocellulose; microbial pretreatment; hydrolysis; biogas. 1
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1. INTRODUCTION
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Water hyacinth is a freshwater lignocellulosic floating weed hampering the aquatic
30
ecosystem, trade and amusement activities of people worldwide due to its incessant
31
reproduction potential (Forrest et al., 2010, Barua and Kalamdhad, 2016). Anaerobic
32
digestion of water hyacinth for the generation of biogas is a sustainable alternative route for
33
the production of eco-friendly energy and management of the weed. Utilisation of renewable
34
biogas as a source of energy possibly will replace fossil fuels, minimise the emission of
35
greenhouse gases, trim down the depletion of resources, reduce the reliance on external
36
sources of energy and cut down cost. Existence of the tightly associated recalcitrant lignin,
37
crystalline cellulose and hemicellulose polymers, delays hydrolysis stage and restrains biogas
38
production during anaerobic digestion. The structural and defensive responsibility undertaken
39
by the three dimensional lignocellulosic weed opposes both microbial and enzymatic attack
40
(Mosier et al., 2005). This three dimensional (3D) lignocellulosic matrix in plants acts as a
41
shield to both abiotic and biotic aggression preventing easy degradation. Pretreatment helps
42
in dissolving the lignin barrier and dislocating the crystalline arrangement of cellulose present
43
in the biomass. Hence, pretreatment of lignocellulosic complex is essential to diminish the
44
biomass recalcitrance. In other words, pretreatment assists in hydrolysing the lignocellulosic
45
polysaccharides into soluble monosaccharides which can be readily used by microbial
46
biocatalysts during anaerobic digestion. Alexandropoulou et al. (2016) stated that the ultimate
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goal of lignocellulose pretreatment is to achieve delignification, sugar solubilisation and
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reduce cellulose crystallisation. Nowadays, pretreatment of lignocellulosic feedstock during
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anaerobic digestion, seems to be an inevitable step. Previous available literatures have stated
50
the use of thermal pretreatment (Barua and Kalamdhad, 2017), thermochemical pretreatment
51
(Lin et al., 2015), electrohydrolysis pretreatment (Barua et al., 2017) of water hyacinth for
52
accelerating hydrolysis period. But, biological (microbial and enzymatic) pretreatment of 2
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water hyacinth for the production of biogas has been rarely studied, may be because of its
54
relatively time consuming process than the other pretreatment techniques. The utilisation of
55
bacterial strains for solubilising water hyacinth is inexpensive when compared to the direct
56
utilisation of enzymes available in the market. Nevertheless, biological pretreatment does not
57
generate any inhibitors (phenolic compounds, furfurals and hydroxymethylfurfural) during
58
anaerobic digestion and is an eco-friendly process with low energy and chemical input.
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Microbial pretreatment is considered to be inexpensive when compared to other
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physicochemical pretreatment methods. Physical pretreatment methods require specialized
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equipments and machineries which lead to abundant energy consumption and chemical
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pretreatment methods involve acid, alkali which in turn creates harsh condition by generating
63
inhibitory compounds (Mosier, 2005) that might hinder the anaerobic digestion process.
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While microbial pretreatment uses metabolites of microorganisms present in nature for
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rupturing the sturdy lignocellulosic structure thereby enhancing biogas generation.
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Microorganisms are capable of continuously disintegrating the complex organic matter
67
during the different phases of growth (Aydin, 2016). Microbial pretreatment technique
68
involves inoculation of bacterial, fungal or a mixed consortium in the lignocellulosic
69
substrate to degrade cellulose or hemicellulose or lignin (Zhong et al., 2016).
70
Microorganisms i.e., brown, white and soft rot fungi (Nkemka et al., 2015, Su et al., 2016)
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and bacteria are mostly employed for microbial pretreatment to attack the lignocellulosic
72
material by secreting their enzymes. There are even many literatures available which state the
73
utililisation of pure culture (Cater et al., 2015) or microbial consortium (Poszytek et al., 2016,
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Lin et al., 2017) pretreatment for anaerobic digestion of lignocellulosic waste material. The
75
microorganisms during biological pretreatment hydrolyse the lignocellulosic substrate
76
generating soluble oligomeric and monomeric compounds that are further converted into
77
volatile fatty acids (VFA) by acidogenic and acetogenic microorganisms. The VFA (i.e.,
3
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acetic acid, butyric acid, propanoic acid) produced during microbial pretreatment are
79
beneficial for biogas generation. Microbial pretreatment when compared to enzymatic
80
pretreatment demonstrates much better outcome in anaerobic digestion process due to their
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higher functional diversity and tolerance to environmental factors i.e., temperature and pH
82
(Shrestha et al., 2017). Kuijk et al. (2015) stated that optimisation of the most effective strain
83
and culture conditions can make the pretreatment process more proficient by dropping the
84
pretreatment time and carbohydrate loss. Factors influencing biological pretreatment are
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biomass composition, nature of microorganism, incubation time and temperature. Profuse
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cellulolytic and hemicellulolytic microorganisms are present in nature which can be
87
employed for effective water hyacinth pretreatment in order to enhance biogas generation.
88
Therefore in this study, cellulolytic microorganisms isolated from soil, the gut of silverfish
89
(Lepisma saccharina) and millipede (Diplopoda) are employed for water hyacinth
90
pretreatment. Syntrophic symbiotic microflora is present in the guts of these insects which aid
91
in the digestion of cellulose. Lepisma saccharina, the scientific name of silverfish, itself
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indicates that the insect’s food source is carbohydrates. They are basically considered as
93
household pests due to their consumption property of paper, books and photographs to name
94
a few. While most of the millipedes and microorganisms present in soil, feed on plant debris.
95
From, microorganisms present in soil and both of the insects’ food habits; it is quite obvious
96
that they are efficient in degrading cellulose. Microorganisms present in nature are capable of
97
depolymerising the lignocellulosic polymers with the aid of the enzyme system available
98
within the gut thereby leading to enhanced hydrolysis of the substrate. In the gut system of
99
these insects, microorganisms secrete hydrolytic enzymes such as cellulase, hemicellulase
100
and lignin degrading enzymes which can easily hydrolyse lignocellulosic complex. Microbial
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pretreatment is regarded as an eco-friendly method for anaerobic digestion of lignocellulosic
102
waste.
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This study involves the utilisation of cellulolytic bacteria which synthesises potent
104
cellulolytic enzymes to solubilise water hyacinth during the hydrolysis phase of anaerobic
105
digestion. Therefore, the present study was conducted to optimise the most effective bacterial
106
strain isolated from soil and both of the insects’ gut for biological pretreatment of water
107
hyacinth. This is a novel study as to the best of knowledge, optimisation of the most efficient
108
microorganism isolated from soil and the gut of lignocellulose consuming insect has been
109
rarely studied for the biological pretreatment of water hyacinth in order to accelerate
110
hydrolysis and enhance biogas production.
111
2. MATERIALS AND METHODS
112
2.1 Raw material
113
Whole water hyacinth plant (substrate) and cow dung was collected from the pond of
114
Amingaon industrial area and Amingaon village, situated near Indian Institute of Technology
115
Guwahati (IITG) campus, India respectively. The collected whole water hyacinth plant was
116
ground in the ratio 13: 69: 45 (i.e., leaves, stem and root) after chopping to maintain the
117
consistency of the sample. The initial characteristics of the substrate (water hyacinth)
118
determined before starting the experiment is incorporated in table 1.
119
Table 1: Initial characteristics of water hyacinth. Parameters
Water hyacinth
pH
5.55±2
Moisture content (%)
92±5
Volatile solids (VS) (%)
64±5
Soluble chemical oxygen demand
2000±50
(sCOD) (mg/L) VFA (mg/L)
750±20
120
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2.2 Microbial pretreatment of water hyacinth The aim of this microbial pretreatment study was to optimise the ideal bacterial strain for
123
enhancing the hydrolysis of water hyacinth during anaerobic digestion. Bacterial strain
124
isolated from soil (UN3d2), silver fish (SFa2) and millipede (BRb2) was cultivated in
125
autoclaved carboxymethyl cellulose (CMC) media for 2 days at 37◦C, 120 rpm. The bacterial
126
culture was ready for inoculation after 2 days of cultivation. The freshly pulverised water
127
hyacinth mixed with minimal salt media (MSM) was inoculated with different dosage of
128
bacterial culture (108, 109 and 1010 CFU/mL). After inoculation, the substrate was kept inside
129
a shaking incubator at 37◦C, 120 rpm. A control sample (only pulverised water hyacinth) was
130
set aside exclusive of microbial pretreatment. Sample analysis was performed on every
131
second day.
132
2.3 Analytical study
133
Analytical study of sCOD (APHA, 2005) and VFA (Dilalo and Albertson, 1961) were
134
performed to determine the optimum condition required for the solubilisation of water
135
hyacinth during microbial pretreatment. 5 g of sample mixture was collected and distilled
136
water was added to make up the volume 100 mL. The diluted sample was placed in a
137
horizontal shaker for 2 h at 150 rpm and filtered. The filtered sample was directly used for
138
analysing sCOD and VFA. Lignin, cellulose (National renewable energy laboratory; NREL
139
procedure, Updegraff, 1969) and hemicellulose analysis were also performed. 0.3 g (dry
140
mass) of sample was utilised to examine the quantity of acid insoluble lignin by gravimetric
141
method. The hydrolysate acquired subsequent to filtration of the cooled insoluble lignin was
142
utilised to determine acid soluble lignin by UV spectrophotometer (205 nm). For analysing
143
cellulose, acetic/nitric acid (3mL) was poured to the 0.5 g of sample and allowed to boil. The
144
centrifuged supernatant was disposed after cooling. Anthrone reagent was poured in the
145
diluted sample and boiled for 10 mins. Later the absorbance was determined at 630 nm. The
6
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residue was rinsed initially with distilled water followed by 67% H2SO4. Hemicellulose was
147
determined by substracting acid detergent fibre (ADF) from neutral detergent fibre (NDF)
148
(Goering and Van, 1975).
149
2.4 Molecular identification
150
The bacterial isolates were outsourced for DNA sequencing and molecular identification.
151
The partial 16s rRNA sequencing for all the isolates were performed in Yaazh Xenomics
152
(Tamil Nadu, India). For bacterial identification, 16S rRNA genes were amplified by PCR
153
using 16S rRNA gene specific primers. The primers employed for UN3d2 are 8F
154
(AGAGTTTGATCCTGGCTCAG) and 1541R (AAGGAGGTGATCCAGCCGCA). For
155
SFa2, 27F (AGAGTTTGATCMTGGCTCAG) and 1492R
156
(TACGGYTACCTTGTTACGACTT) were utilised whereas for BRb2 the primers employed
157
were 27F (AGAGTTTGATCMTGGCTCAG) and 1492R
158
(TACGGYTACCTTGTTACGACTT). Clone sequences were matched with the GenBank
159
nucleotide database via BLAST microbial genomes searches
160
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) to detect their closest phylogenetic neighbours.
161
2.5 Anaerobic biodegradability test
162
Anaerobic biodegradability test or the biochemical methane potential (BMP) test was
163
carried out in batch mode between the optimised bacterial strain illustrating highest
164
solubilisation of water hyacinth and untreated water hyacinth. Tests were performed in
165
triplicate for a food to microorganism (F/M) ratio of 1.5:1 determined on the basis of VS
166
content of the water hyacinth and the cow dung. The 1 L reactor bottles were fed with
167
pretreated and untreated pulverised water hyacinth, cow dung and with MSM. The level of
168
the reactor bottles were maintained by filling distilled water. Then nitrogen gas was purged
169
into the reactor bottles to retain anaerobic situation, tightly shut and were connected to the1.5
170
N NaOH containing aspirator bottles (Elliott and Mahmood, 2007). Biogas generation was 7
ACCEPTED MANUSCRIPT 171
measured daily by liquid displacement method. The analysis was performed for 50 days.
172
Once the 1 L BMP study was over, the batch study was scaled up to 20 L to test the
173
operational conditions during large scale and to decrease the unevenness due to
174
heterogeneous physico-chemical characteristics of water hyacinth. The 20 L batch study was
175
conducted for 80 days.
176
2.6 Kinetic study
177
Modified Gompertz equation was utilised to estimate the optimum factors required for the
178
activity of methanogens to generate maximum biogas. Modified Gompertz equation assumes
179
that the rate of biogas generation is directly proportional to the activity of microorganisms
180
inside the anaerobic reactor.
181
𝑌 = 𝑀 × 𝑒𝑥𝑝 ‒ 𝑒𝑥𝑝
{
[
𝑅𝑚 × 𝑒 𝑀
]} ....... (1)
(𝜆 ‒ 1) + 1
182 183
Where Y correspond to the cumulative methane generation (mL) at time t (d), M is the
184
maximum methane generation potential (mL CH4), Rm is the maximum methane generation
185
rate (mL CH4 d-1), λ is the lag phase time (d) and e is constant equivalent to 2.71. The
186
parameters M, Rm and 𝜆 were determined through curve-fitting via Matlab R2015b by
187
reducing the residual quantity of squared ambiguity between the experimental statistics and
188
the modeled curve.
189 190
3. RESULTS AND DISCUSSION 3.1 Effect of microbial (bacterial) pretreatment on solubilisation of the water hyacinth
191
In order to rupture the lignocellulosic cell wall and to improve the solubilisation of the
192
substrate, isolated microorganisms were employed to pretreat water hyacinth. With respect to
193
the solubilisation process of water hyacinth an appropriate selection of bacterial strain/
194
culture is necessary. Therefore to determine the ideal isolated bacterial culture providing the
195
highest solubilisation of water hyacinth, sCOD and the VFA of the samples were analysed at 8
ACCEPTED MANUSCRIPT 196
various dosage and time. Fig 1a, 1b, 2a, 2b, 3a and 3b depicts similar trends of increase in
197
sCOD and VFA concentration. sCOD and VFA increased hand in hand as the number of days
198
increased and after reaching the utmost peak, it starts decreasing. The increase in VFA led to
199
the increase in sCOD. For water hyacinth, pretreatment with SFa2 illustrated the highest
200
sCOD (solubilisation) in 4 days followed by BRb2 and UN3d2 (6 days) at a dosage of 109
201
CFU/mL. Although the same dose of inoculum showed the highest solubilisation for the
202
entire three different bacterial strains still the degree of solubilisation and the time required
203
for solubilisation differed. SFa2 illustrated the highest solubilisation of 1.33 times or 33.33%
204
followed by BRb2 (1.26 times or 26.75%) and UN3d2 (1.21 times or 20.94%). The release of
205
extracellular and intracellular biopolymers into the soluble phase is a fundamental factor to
206
assess the proficiency of the pretreatment (Mahdy et al., 2016). Initially, the sCOD and VFA
207
for water hyacinth inoculated with 109 CFU/mL of SFa2 was observed to be 2496 mg/L and
208
764 mg/L respectively. As the time required for solubilising is one of the key administering
209
feature in bacterial pretreatment (Kavitha et al., 2017), the release of soluble organic matter
210
amplifies with an augment in pretreatment time. On the 2nd day of pretreatment, sCOD and
211
VFA enhanced to 2506 mg/L and 1000 mg/L, indicating that the rise in sCOD and VFA may
212
be attributable to exopolymeric matrix discharge rather than the availability of the
213
intracellular polymers. More increment in the pretreatment time to 4 days displayed a radical
214
increase in sCOD and VFA to 4160 mg/L and 1480 mg/L respectively. This radical increase
215
in sCOD designates the secretion of cellulase enzyme by the SFa2 bacteria. Firstly, because
216
the generated compounds are the substrates, as a result the recalcitrant lignocellulosic cell
217
wall complex ruptured resulting in the discharge of extracellular and intracellular polymers
218
into the soluble phase. From the 6th day, both VFA and sCOD started declining. Yuan et al.
219
(2014) demonstrated rapid increase in the concentration of VFA and sCOD during 4 days of
220
microbial pretreatment of corn stalk. High sCOD indicates that huge amount of soluble
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organic matter are present for anaerobic digestion and the formed VFA performs a significant
222
responsibility in accelerating the hydrolysis of lignocellulosic material (Yu et al., 2004). The
223
mode of action of the microbial pretreatment can be hypothesised by two credible
224
mechanisms. First of all, the bacteria might have attacked the sturdy outer layer by
225
dissolving/ softening down the lignin resilience thereby increasing the permeability of the
226
water hyacinth. Followed by the second mechanism of the bacterial attack in the inner layer
227
of the water hyacinth and finally leading to the hydrolysis of the cellulose and hemicellulose.
228
The bacteria initially starts consuming the easily available soluble organic matter; once the
229
soluble organic matter is over, the bacteria starts secreting exoenzymes to solubilise the
230
particulate organic matter thereby increasing the quantity of soluble organic matter and the
231
bacterial population, leading to the increase in sCOD. After few days, as the availability of
232
food decreases, the bacteria starts consuming each other, leading to the decrease in sCOD.
233
The outcome designates that the conversion of complex organic matter to soluble organic
234
matter can be time consuming, and the optimum pretreatment time and dosage must be
235
equivalent to that, during which sCOD and the VFA accomplishes the highest peak value.
236
The increased level of VFA and sCOD are a proof of the high hydrolytic and cellulolytic
237
activity of the isolated bacteria especially SFa2. Thus the optimal pretreatment dosage and
238
time is 109 CFU/mL of SFa2 in 4 days for water hyacinth. This optimal pretreatment dosage
239
and time makes sure that the exhaustion of soluble organic matter during microbial
240
pretreatment is reduced and that the soluble organic matter is available for biogas generation.
241
3.2 Effect of microbial pretreatment on the compositional analysis of water hyacinth
242
The main objective of the microbial pretreatment is to rupture the lignocellulosic complex
243
of water hyacinth so that a higher amount of easily degradable organic carbon fraction is bio-
244
accessible. Improved biodegradability of water hyacinth can lead to increased biogas
245
production in a short duration as the recalcitrant lignin softens making it easier for the
10
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microorganisms to feed on the depolymerised cellulose. Therefore compositional analysis
247
was carried out to study the modification in the lignocellulosic composition of the water
248
hyacinth before and after microbial pretreatment (Table 2).
249
Lignin is a complex, aromatic heteropolymer in which its main structural constituents,
250
phenylpropanoid aryl-C3 units, are connected by a diverse range of C-O and C-C
251
bonds. Cellulose is a polysaccharide, composed of a linear string of D-glucose molecules
252
connected by β-(1, 4)-glycosidic bonds. Hemicellulose is an easily hydrolysable branched
253
heterogeneous polymer of pentose (xylose, arabinose), hexose (mannose, glucose and
254
galactose) and acetylated sugars. Enhanced delignification in the pretreated substrate when
255
compared to the untreated was evident as the percentage of soluble lignin increased. SFa2
256
pretreated sample substantiates the utmost increase in the percentage of soluble lignin
257
(3.23±0.8 %) followed by BRb2 (2.93±0.1 %) and UN3d2 (2.82±0.4 %) pretreated when
258
compared to the untreated substrate (2.77±0.3 %). The presence of more soluble lignin
259
signifies the softening of the sturdy lignin. The softened lignin makes the cellulose easily bio-
260
accessible. Repolymerization reaction of the lignin is the main reason behind the slight
261
increase in the percentage of acid insoluble lignin in the pretreated samples (Li et al., 2007).
262
Reduction in the composition of cellulose in pretreated water hyacinth than the untreated is
263
advantageous as it represents enhanced solubilisation. The long string cellulose must have
264
split at some indefinite point by the microorganisms during pretreatment leading to the
265
decline in cellulose (Barua and Kalamdhad, 2017). In this manner the percentage of short
266
string glucose fragments (hemicellulose) in the microbial pretreated water hyacinth increased.
267
Highest reduction in cellulose was observed in SFa2 (30.89±0.2 %) followed by BRb2
268
(32.63±0.3 %) and UN3d2 (33.87±0.8 %) respectively. Conversely, SFa2 pretreated water
269
hyacinth illustrated the increase in the availability of hemicellulose (43.06±0.7 %) than the
270
untreated water hyacinth (27.7±0.2 %). Microbial pretreatment helped in the modification of
11
ACCEPTED MANUSCRIPT 271
the chemical composition and physical configuration of the lignocellulosic water hyacinth.
272
These chemical and physical modifications team up to improve the biodegradability of the
273
water hyacinth and enhance biogas generation. Thus the compositional analysis indicates the
274
availability of the utmost amount of readily degradable soluble organics in the SFa2
275
pretreated water hyacinth.
276
Table 2: Compositional changes in water hyacinth due to microbial pretreatment Microbial
Acid
soluble Acid
insoluble Cellulose (%)
Hemicellulose
consortium
lignin (%)
lignin (%)
--
2.77±0.3
7.93±0.5
36.84±0.8
27.7±0.2
SFa2
3.23±0.8
8.34±0.3
30.89±0.2
43.06±0.7
BRb2
2.93±0.1
8.92±0.6
32.63±0.3
36.02±0.8
UN3d2
2.82±0.4
9.05±0.9
33.87±0.8
30.62±0.9
(%)
277 278
3.3 Molecular identification
279
All the three bacterial isolates i.e., SFa2, BRb2 and UN3d2 were identified
280
phylogenetically (Table 3). 16S rRNA analysis helped in the identification of the naturally
281
isolated three bacterial strains. 16S rRNA analysis technique assists in recognising poorly
282
described or rarely isolated strains thereby leading to the recognition of the isolated novel
283
bacterial strains. 16S rRNA gene sequences analysis is also useful for determining the extent
284
of phylogenetic diversity within diverse groups of bacteria. The phylogenetic tree or
285
dendogram represents the evolutionary relationships between various biological organisms.
286
The phylogenetic analysis (fig 4a) revealed SFa2 to be of the phylum Proteobacteria, which is
287
a major phylum of gram negative bacteria. The outer membrane of Proteobacteria is
288
composed of lipopolysaccharides. The identified Proteobacteria was indicated as Citrobacter
289
werkmanii. The nucleotide sequence of the bacterial strain SFa2 displayed 91 % sequence 12
ACCEPTED MANUSCRIPT 290
identity with their closest relatives. Accession number MF099900 was provided by GenBank
291
for SFa2. UN3d2 (fig 4c) also belonged to the phylum Proteobacteria and was identified as
292
Bordetella muralis with an accession number MF098756. While BRb2 (fig 4b) was a
293
member belonging to the phylum Firmicutes. BRb2 was identified as Paenibacillus sp with
294
an accession number MF187530. An accession number is a unique identifier provided to a
295
DNA or protein sequence record for identifying dissimilar versions of that sequence record
296
and the related sequence over time in a solo information repository. Citrobacter werkmanii
297
(MF099900) pretreatment illustrated the highest solubilisation of water hyacinth. Thereby
298
indicating that Citrobacter werkmanii VKVVG4 will be most beneficial in accelerating the
299
hydrolysis of water hyacinth and enhancing the generation of biogas when compared to the
300
other bacterial isolates. Table 3: Phylogenetic identification of the isolates.
301 302 303
Code
Closest relative
Accession
% Solubilisation
number SFa2
Citrobacter werkmanii
MF099900
33.3
MF187530
26.75
MF098756
20.94
VKVVG4
BRb2
Paenibacillus sp VKVVG1
UN3d2
Bordetella
muralis
VKVVG5
304
3.4 Effect of microbial pretreatment on biogas production
305
BMP test was performed between untreated and microbial (SFa2) pretreated water
306
hyacinth as SFa2 illustrated 1.33 times increase in solubilisation than the untreated water 13
ACCEPTED MANUSCRIPT 307
hyacinth. Freshly pulverised water hyacinth whole plant was inoculated with 109 CFU/mL of
308
SFa2 and was incubated at 37◦C, 150 rpm for 4 days. After 4 days of pretreatment, cow dung
309
was added to the pretreated substrate and employed for BMP study. MSM was provided to
310
supply the necessary macro and micro nutrients to the microorganisms.
311
During the initial week of the anaerobic digestion process, lag phase was observed for
312
both the untreated and microbial (SFa2) pretreated water hyacinth due to the acclimatisation
313
phase of the cow dung with the substrate environment inside the anaerobic reactor (fig 5).
314
After a week of anaerobic digestion, biogas production begins to increase with the increase in
315
anaerobic digestion period. A sharp increase in the cumulative biogas production curve for
316
the SFa2 pretreated water hyacinth was observed. Hence, indicating that SFa2 pretreatment
317
was competent enough to hydrolyse some amount of the complex organic macromolecules
318
available in the water hyacinth by forming more readily biodegradable substances available
319
for anaerobic digestion. Due to augmentation of biogas production rate, the cumulative
320
biogas production amplified incessantly during the digestion period. Significant increase in
321
biogas production was observed during the log phase on account of the rapid multiplication
322
and speedy consumption of the water hyacinth by the methanogens. Further increase in
323
biogas production persisted throughout the log and steady phase. SFa2 pretreated water
324
hyacinth achieved a cumulative biogas production of 2123±10 mL on the 20th day whereas
325
the untreated water hyacinth achieved 1023±17 mL of cumulative biogas on the same day.
326
By the end of 50 days, SFa2 pretreated water hyacinth illustrated a cumulative biogas
327
production of 3737±21 mL whereas untreated water hyacinth illustrated a cumulative biogas
328
production of 3038±13 mL. The increased biogas production from water hyacinth after
329
microbial pretreatment signifies the presence of easily digestible soluble organic matter. The
330
enhanced solubilisation of water hyacinth after microbial pretreatment led to an increase in
331
biogas production. Cordoba et al., 2016 reported a cumulative biogas production of
14
ACCEPTED MANUSCRIPT 332
3960 ± 431 mL in 105 days from spent saw dust after microbial (Gymnopilus pampeanus)
333
pretreatment. There are many previous literatures which states of witnessing the increase in
334
biogas production after biological pretreatment of corn straw and saw dust waste respectively
335
(Zhong et al., 2011; Ali et al., 2017). Hydrolysis of untreated water hyacinth is a slow process
336
and yields less biogas when compared to the SFa2 pretreated water hyacinth. Microbial
337
pretreatment immensely influences the rate of the anaerobic digestion process and
338
biodegradability of the water hyacinth. This highlights the fact that the discharge of soluble
339
organic matter during microbial pretreatment helps in the biodegradability of the substrate,
340
enhancing biogas yield and process kinetics. Microbial (SFa2) pretreatment provoked desired
341
structural and chemical modification in the lignocellulosic water hyacinth, thereby enhancing
342
the biomethanation process. As, the lignocellulosic complex of the untreated water hyacinth
343
was tightly intact, slow hydrolysis period was witnessed. In other words the presence of
344
lignin barrier and crystalline long cellulosic fibres in the untreated water hyacinth hindered
345
the microorganisms to work synergistically, ultimately producing lesser quantity of biogas.
346
Sturdy lignin and crystalline cellulose requires more time to disintegrate into labile carbon
347
moieties. Thus, microbial pretreatment was adequately efficient for rapid biodegradation of
348
the water hyacinth leading to faster hydrolysis and enhanced biogas production.
349
3.5 Scaled up batch anaerobic digestion and its kinetics
350
As all the above analytical studies and biogas production studies demonstrated positive
351
results therefore the batch study was scaled up to 20 L in order to check the operational
352
conditions and variations to be encountered during scaled up process.
353
From Fig 7, the massive increase in biogas production from water hyacinth after
354
microbial pretreatment is convincingly evident. Cumulative biogas production from water
355
hyacinth after microbial pretreatment was observed to be 102491±13 mL in 80 days whereas
356
a cumulative biogas production of 50612±18 mL only was obtained from untreated water 15
ACCEPTED MANUSCRIPT 357
hyacinth for the same number of days. An increase of 3.07 times in biogas production was
358
observed in the 20 L batch study. The highest daily biogas production of 4898±10 mL was
359
achieved on 21st day after microbial pretreatment of water hyacinth. Attaining the highest
360
daily biogas production on 21st day was quite faster than the untreated water hyacinth. The
361
highest daily biogas production of untreated water hyacinth was achieved on the 28th day.
362
This indicates the acceleration in the hydrolysis phase of water hyacinth after microbial
363
pretreatment. Even the amount of highest daily biogas production from the untreated water
364
hyacinth (2016±13 mL) on the 28th day was far lesser when compared to the highest daily
365
biogas production of 4898±10 mL achieved on 21st day after microbial pretreatment of water
366
hyacinth. A dynamic change in the generation of biogas was witnessed in the water hyacinth
367
after microbial pretreatment. The enhanced amount of biogas production from water hyacinth
368
after microbial pretreatment denotes the presence of excessive amount of easily soluble
369
compounds. In the untreated water hyacinth due to the presence of recalcitrant lignin and
370
crystalline cellulose, the activity of the microorganisms was slow. The effective strain and
371
ideal culture condition made the pretreatment process efficient by reducing the hydrolysis
372
period almost by a week and by enhancing biogas production.
373
The acquired estimated kinetic values are provided in table 4. The values signifies that
374
the modified Gompertz equation can be employed to estimate the maximum methane
375
generation potential, the maximum methane generation rate and the lag phase. The kinetic
376
parameters of the untreated and microbial pretreated water hyacinth utilised during 20 L
377
batch study was determined where the values of M of the pretreated water hyacinth (171.039
378
L CH4) was proved to be better than the untreated water hyacinth (71. 796 L CH4). R2 value
379
surpassed 0.90 for both untreated and microbial pretreated water hyacinth, suggesting that
380
methane generation can be well simulated. Thus, the curve of the modified Gompertz
381
equation fitted well with the experimental values of cumulative biogas generation (Fig 6). 16
ACCEPTED MANUSCRIPT 382
Table 4: Kinetics values of untreated and microbial pretreated water hyacinth used in 20 L
383
batch study. Substrate
M
Rmax
λ (d)
(L CH4)
(L CH4 d-
R2
Y (L CH4)
1)
Pretreated
171.039
0.1000
0.0000
0.96
102.49
Untreated
71. 796
0.0772
0.0010
0.92
50.65
384 385
386
4. CONCLUSION Microbial pretreatment of water hyacinth proved to be proficient in accelerating the
387
hydrolysis step of anaerobic digestion by improving the biodegradability of the substrate.
388
Pretreatment of water hyacinth with SFa2 (Citrobacter werkmanii VKVVG4, MF099900)
389
helped in achieving the highest solubilisation of 33.33% with an optimum dosage of 109
390
CFU/mL within 4 days. Both BMP (1 L) and batch (20 L) assay revealed that the cumulative
391
methane production of SFa2 pretreated water hyacinth was higher than the untreated water
392
hyacinth. 1 L BMP assay illustrated that SFa2 pretreated water hyacinth attained a cumulative
393
biogas production of 3737±21 mL whereas untreated water hyacinth attained a cumulative
394
biogas production of 3038±13 mL on the 50th day. Thus, Citrobacter werkmanii VKVVG4 or
395
SFa2 pretreatment of water hyacinth succeeded in enhancing biogas production also.
396
Acknowledgement
397 398
The authors are grateful to Mr. Vikas Kumar, Centre for Energy, Indian Institute of Technology Guwahati, Assam, India.
399 17
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Fig 1: Changes in (a) sCOD and (b) VFA concentration during microbial pretreatment with SFa2.
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ACCEPTED MANUSCRIPT (a) 4000
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Fig 3: Changes in (a) sCOD and (b) VFA concentration during microbial pretreatment with UN3d2
ACCEPTED MANUSCRIPT Cumulative biogas produced (mL)
4500 untreated
4000
pretreated
3500 3000 2500 2000 1500 1000 500 0 0
5
10Time15(Days) 20
25
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35
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Fig 4: Cumulative biogas production of untreated and pretreated water hyacinth. (a)
(b)
(c)
Fig 5: Phylogenetic tree based on 16S rRNA gene sequences of bacterial strain (a) SFa2 (Citrobacter werkmanii VKVVG4) (b) BrB2 (Paenibacillus sp. VKVVG1) and (c) Un3d2 (Bordetella muralis VKVVG5)
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Cumulative Biogas Produced (mL)
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ACCEPTED MANUSCRIPT HIGHLIGHTS
Novel bacterial pure cultures were utilized to pretreat water hyacinth. Microbial pretreatment showed enhanced solubilisation of water hyacinth. Citrobacter werkmanii VKVVG4 illustrated highest solubilisation of water hyacinth. Microbial pretreatment improved biogas production from water hyacinth.
Visva Bharati Barua, Vaibhav V. Goud, Ajay S. Kalamdhad PII:
S0960-1481(18)30334-3
DOI:
10.1016/j.renene.2018.03.028
Reference:
RENE 9897
To appear in:
Renewable Energy
Received Date:
07 October 2017
Revised Date:
04 January 2018
Accepted Date:
13 March 2018
Please cite this article as: Visva Bharati Barua, Vaibhav V. Goud, Ajay S. Kalamdhad, Microbial Pretreatment of Water Hyacinth for Enhanced Hydrolysis followed by Biogas Production, Renewable Energy (2018), doi: 10.1016/j.renene.2018.03.028
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
2
Microbial Pretreatment of Water Hyacinth for Enhanced Hydrolysis followed by Biogas Production
3
Visva Bharati Baruaa,*, Vaibhav V. Goudb and Ajay S. Kalamdhadc
4 5
aCentre
6 7
bDepartment
8 9
cDepartment
1
for the Environment, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India of Chemical Engineering, Indian Institute of Technology Guwahati, Guwahati781039, Assam, India of Civil Engineering, Indian Institute of Technology Guwahati, Guwahati781039, Assam, India
10
*email: [email protected]
11
ABSTRACT
12
Biological pretreatment with novel isolated microbial pure culture was utilised to pretreat
13
water hyacinth to enhance its solubilisatio n followed by biogas production. Lignocellulose
14
degrading bacterial strains isolated from soil (Bordetella muralis VKVVG5) (UN3d2), the gut
15
of silverfish (Citrobacter werkmanii VKVVG4) (SFa2) and millipede (Paenibacillus sp.
16
VKVVG1) (BrB2) were employed to optimise the ideal bacterial strain illustrating accelerated
17
hydrolysis of water hyacinth. Citrobacter werkmanii VKVVG4 pretreatment of water hyacinth
18
with an optimum dosage of 109 CFU/mL and time of 4 days helped in achieving the highest
19
solubilisation of 33.3%. Biochemical methane potential (BMP) test was conducted between
20
untreated and Citrobacter werkmanii VKVVG4 pretreated water hyacinth. Biochemical
21
methane potential (BMP) test of pretreated water hyacinth illustrated faster start up period
22
than the untreated water hyacinth. Citrobacter werkmanii VKVVG4 (SFa2) pretreated water
23
hyacinth illustrated a cumulative biogas production of 3737±21 mL whereas untreated water
24
hyacinth illustrated a cumulative biogas production of 3038±13 mL on the 50th day. Scaled
25
up batch (20 L) study demonstrated a three times increase in the cumulative biogas
26
generation of the microbial pretreated water hyacinth than the untreated water hyacinth.
27
Keywords: Water hyacinth; lignocellulose; microbial pretreatment; hydrolysis; biogas. 1
ACCEPTED MANUSCRIPT 28
1. INTRODUCTION
29
Water hyacinth is a freshwater lignocellulosic floating weed hampering the aquatic
30
ecosystem, trade and amusement activities of people worldwide due to its incessant
31
reproduction potential (Forrest et al., 2010, Barua and Kalamdhad, 2016). Anaerobic
32
digestion of water hyacinth for the generation of biogas is a sustainable alternative route for
33
the production of eco-friendly energy and management of the weed. Utilisation of renewable
34
biogas as a source of energy possibly will replace fossil fuels, minimise the emission of
35
greenhouse gases, trim down the depletion of resources, reduce the reliance on external
36
sources of energy and cut down cost. Existence of the tightly associated recalcitrant lignin,
37
crystalline cellulose and hemicellulose polymers, delays hydrolysis stage and restrains biogas
38
production during anaerobic digestion. The structural and defensive responsibility undertaken
39
by the three dimensional lignocellulosic weed opposes both microbial and enzymatic attack
40
(Mosier et al., 2005). This three dimensional (3D) lignocellulosic matrix in plants acts as a
41
shield to both abiotic and biotic aggression preventing easy degradation. Pretreatment helps
42
in dissolving the lignin barrier and dislocating the crystalline arrangement of cellulose present
43
in the biomass. Hence, pretreatment of lignocellulosic complex is essential to diminish the
44
biomass recalcitrance. In other words, pretreatment assists in hydrolysing the lignocellulosic
45
polysaccharides into soluble monosaccharides which can be readily used by microbial
46
biocatalysts during anaerobic digestion. Alexandropoulou et al. (2016) stated that the ultimate
47
goal of lignocellulose pretreatment is to achieve delignification, sugar solubilisation and
48
reduce cellulose crystallisation. Nowadays, pretreatment of lignocellulosic feedstock during
49
anaerobic digestion, seems to be an inevitable step. Previous available literatures have stated
50
the use of thermal pretreatment (Barua and Kalamdhad, 2017), thermochemical pretreatment
51
(Lin et al., 2015), electrohydrolysis pretreatment (Barua et al., 2017) of water hyacinth for
52
accelerating hydrolysis period. But, biological (microbial and enzymatic) pretreatment of 2
ACCEPTED MANUSCRIPT 53
water hyacinth for the production of biogas has been rarely studied, may be because of its
54
relatively time consuming process than the other pretreatment techniques. The utilisation of
55
bacterial strains for solubilising water hyacinth is inexpensive when compared to the direct
56
utilisation of enzymes available in the market. Nevertheless, biological pretreatment does not
57
generate any inhibitors (phenolic compounds, furfurals and hydroxymethylfurfural) during
58
anaerobic digestion and is an eco-friendly process with low energy and chemical input.
59
Microbial pretreatment is considered to be inexpensive when compared to other
60
physicochemical pretreatment methods. Physical pretreatment methods require specialized
61
equipments and machineries which lead to abundant energy consumption and chemical
62
pretreatment methods involve acid, alkali which in turn creates harsh condition by generating
63
inhibitory compounds (Mosier, 2005) that might hinder the anaerobic digestion process.
64
While microbial pretreatment uses metabolites of microorganisms present in nature for
65
rupturing the sturdy lignocellulosic structure thereby enhancing biogas generation.
66
Microorganisms are capable of continuously disintegrating the complex organic matter
67
during the different phases of growth (Aydin, 2016). Microbial pretreatment technique
68
involves inoculation of bacterial, fungal or a mixed consortium in the lignocellulosic
69
substrate to degrade cellulose or hemicellulose or lignin (Zhong et al., 2016).
70
Microorganisms i.e., brown, white and soft rot fungi (Nkemka et al., 2015, Su et al., 2016)
71
and bacteria are mostly employed for microbial pretreatment to attack the lignocellulosic
72
material by secreting their enzymes. There are even many literatures available which state the
73
utililisation of pure culture (Cater et al., 2015) or microbial consortium (Poszytek et al., 2016,
74
Lin et al., 2017) pretreatment for anaerobic digestion of lignocellulosic waste material. The
75
microorganisms during biological pretreatment hydrolyse the lignocellulosic substrate
76
generating soluble oligomeric and monomeric compounds that are further converted into
77
volatile fatty acids (VFA) by acidogenic and acetogenic microorganisms. The VFA (i.e.,
3
ACCEPTED MANUSCRIPT 78
acetic acid, butyric acid, propanoic acid) produced during microbial pretreatment are
79
beneficial for biogas generation. Microbial pretreatment when compared to enzymatic
80
pretreatment demonstrates much better outcome in anaerobic digestion process due to their
81
higher functional diversity and tolerance to environmental factors i.e., temperature and pH
82
(Shrestha et al., 2017). Kuijk et al. (2015) stated that optimisation of the most effective strain
83
and culture conditions can make the pretreatment process more proficient by dropping the
84
pretreatment time and carbohydrate loss. Factors influencing biological pretreatment are
85
biomass composition, nature of microorganism, incubation time and temperature. Profuse
86
cellulolytic and hemicellulolytic microorganisms are present in nature which can be
87
employed for effective water hyacinth pretreatment in order to enhance biogas generation.
88
Therefore in this study, cellulolytic microorganisms isolated from soil, the gut of silverfish
89
(Lepisma saccharina) and millipede (Diplopoda) are employed for water hyacinth
90
pretreatment. Syntrophic symbiotic microflora is present in the guts of these insects which aid
91
in the digestion of cellulose. Lepisma saccharina, the scientific name of silverfish, itself
92
indicates that the insect’s food source is carbohydrates. They are basically considered as
93
household pests due to their consumption property of paper, books and photographs to name
94
a few. While most of the millipedes and microorganisms present in soil, feed on plant debris.
95
From, microorganisms present in soil and both of the insects’ food habits; it is quite obvious
96
that they are efficient in degrading cellulose. Microorganisms present in nature are capable of
97
depolymerising the lignocellulosic polymers with the aid of the enzyme system available
98
within the gut thereby leading to enhanced hydrolysis of the substrate. In the gut system of
99
these insects, microorganisms secrete hydrolytic enzymes such as cellulase, hemicellulase
100
and lignin degrading enzymes which can easily hydrolyse lignocellulosic complex. Microbial
101
pretreatment is regarded as an eco-friendly method for anaerobic digestion of lignocellulosic
102
waste.
4
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This study involves the utilisation of cellulolytic bacteria which synthesises potent
104
cellulolytic enzymes to solubilise water hyacinth during the hydrolysis phase of anaerobic
105
digestion. Therefore, the present study was conducted to optimise the most effective bacterial
106
strain isolated from soil and both of the insects’ gut for biological pretreatment of water
107
hyacinth. This is a novel study as to the best of knowledge, optimisation of the most efficient
108
microorganism isolated from soil and the gut of lignocellulose consuming insect has been
109
rarely studied for the biological pretreatment of water hyacinth in order to accelerate
110
hydrolysis and enhance biogas production.
111
2. MATERIALS AND METHODS
112
2.1 Raw material
113
Whole water hyacinth plant (substrate) and cow dung was collected from the pond of
114
Amingaon industrial area and Amingaon village, situated near Indian Institute of Technology
115
Guwahati (IITG) campus, India respectively. The collected whole water hyacinth plant was
116
ground in the ratio 13: 69: 45 (i.e., leaves, stem and root) after chopping to maintain the
117
consistency of the sample. The initial characteristics of the substrate (water hyacinth)
118
determined before starting the experiment is incorporated in table 1.
119
Table 1: Initial characteristics of water hyacinth. Parameters
Water hyacinth
pH
5.55±2
Moisture content (%)
92±5
Volatile solids (VS) (%)
64±5
Soluble chemical oxygen demand
2000±50
(sCOD) (mg/L) VFA (mg/L)
750±20
120
5
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2.2 Microbial pretreatment of water hyacinth The aim of this microbial pretreatment study was to optimise the ideal bacterial strain for
123
enhancing the hydrolysis of water hyacinth during anaerobic digestion. Bacterial strain
124
isolated from soil (UN3d2), silver fish (SFa2) and millipede (BRb2) was cultivated in
125
autoclaved carboxymethyl cellulose (CMC) media for 2 days at 37◦C, 120 rpm. The bacterial
126
culture was ready for inoculation after 2 days of cultivation. The freshly pulverised water
127
hyacinth mixed with minimal salt media (MSM) was inoculated with different dosage of
128
bacterial culture (108, 109 and 1010 CFU/mL). After inoculation, the substrate was kept inside
129
a shaking incubator at 37◦C, 120 rpm. A control sample (only pulverised water hyacinth) was
130
set aside exclusive of microbial pretreatment. Sample analysis was performed on every
131
second day.
132
2.3 Analytical study
133
Analytical study of sCOD (APHA, 2005) and VFA (Dilalo and Albertson, 1961) were
134
performed to determine the optimum condition required for the solubilisation of water
135
hyacinth during microbial pretreatment. 5 g of sample mixture was collected and distilled
136
water was added to make up the volume 100 mL. The diluted sample was placed in a
137
horizontal shaker for 2 h at 150 rpm and filtered. The filtered sample was directly used for
138
analysing sCOD and VFA. Lignin, cellulose (National renewable energy laboratory; NREL
139
procedure, Updegraff, 1969) and hemicellulose analysis were also performed. 0.3 g (dry
140
mass) of sample was utilised to examine the quantity of acid insoluble lignin by gravimetric
141
method. The hydrolysate acquired subsequent to filtration of the cooled insoluble lignin was
142
utilised to determine acid soluble lignin by UV spectrophotometer (205 nm). For analysing
143
cellulose, acetic/nitric acid (3mL) was poured to the 0.5 g of sample and allowed to boil. The
144
centrifuged supernatant was disposed after cooling. Anthrone reagent was poured in the
145
diluted sample and boiled for 10 mins. Later the absorbance was determined at 630 nm. The
6
ACCEPTED MANUSCRIPT 146
residue was rinsed initially with distilled water followed by 67% H2SO4. Hemicellulose was
147
determined by substracting acid detergent fibre (ADF) from neutral detergent fibre (NDF)
148
(Goering and Van, 1975).
149
2.4 Molecular identification
150
The bacterial isolates were outsourced for DNA sequencing and molecular identification.
151
The partial 16s rRNA sequencing for all the isolates were performed in Yaazh Xenomics
152
(Tamil Nadu, India). For bacterial identification, 16S rRNA genes were amplified by PCR
153
using 16S rRNA gene specific primers. The primers employed for UN3d2 are 8F
154
(AGAGTTTGATCCTGGCTCAG) and 1541R (AAGGAGGTGATCCAGCCGCA). For
155
SFa2, 27F (AGAGTTTGATCMTGGCTCAG) and 1492R
156
(TACGGYTACCTTGTTACGACTT) were utilised whereas for BRb2 the primers employed
157
were 27F (AGAGTTTGATCMTGGCTCAG) and 1492R
158
(TACGGYTACCTTGTTACGACTT). Clone sequences were matched with the GenBank
159
nucleotide database via BLAST microbial genomes searches
160
(http://blast.ncbi.nlm.nih.gov/Blast.cgi) to detect their closest phylogenetic neighbours.
161
2.5 Anaerobic biodegradability test
162
Anaerobic biodegradability test or the biochemical methane potential (BMP) test was
163
carried out in batch mode between the optimised bacterial strain illustrating highest
164
solubilisation of water hyacinth and untreated water hyacinth. Tests were performed in
165
triplicate for a food to microorganism (F/M) ratio of 1.5:1 determined on the basis of VS
166
content of the water hyacinth and the cow dung. The 1 L reactor bottles were fed with
167
pretreated and untreated pulverised water hyacinth, cow dung and with MSM. The level of
168
the reactor bottles were maintained by filling distilled water. Then nitrogen gas was purged
169
into the reactor bottles to retain anaerobic situation, tightly shut and were connected to the1.5
170
N NaOH containing aspirator bottles (Elliott and Mahmood, 2007). Biogas generation was 7
ACCEPTED MANUSCRIPT 171
measured daily by liquid displacement method. The analysis was performed for 50 days.
172
Once the 1 L BMP study was over, the batch study was scaled up to 20 L to test the
173
operational conditions during large scale and to decrease the unevenness due to
174
heterogeneous physico-chemical characteristics of water hyacinth. The 20 L batch study was
175
conducted for 80 days.
176
2.6 Kinetic study
177
Modified Gompertz equation was utilised to estimate the optimum factors required for the
178
activity of methanogens to generate maximum biogas. Modified Gompertz equation assumes
179
that the rate of biogas generation is directly proportional to the activity of microorganisms
180
inside the anaerobic reactor.
181
𝑌 = 𝑀 × 𝑒𝑥𝑝 ‒ 𝑒𝑥𝑝
{
[
𝑅𝑚 × 𝑒 𝑀
]} ....... (1)
(𝜆 ‒ 1) + 1
182 183
Where Y correspond to the cumulative methane generation (mL) at time t (d), M is the
184
maximum methane generation potential (mL CH4), Rm is the maximum methane generation
185
rate (mL CH4 d-1), λ is the lag phase time (d) and e is constant equivalent to 2.71. The
186
parameters M, Rm and 𝜆 were determined through curve-fitting via Matlab R2015b by
187
reducing the residual quantity of squared ambiguity between the experimental statistics and
188
the modeled curve.
189 190
3. RESULTS AND DISCUSSION 3.1 Effect of microbial (bacterial) pretreatment on solubilisation of the water hyacinth
191
In order to rupture the lignocellulosic cell wall and to improve the solubilisation of the
192
substrate, isolated microorganisms were employed to pretreat water hyacinth. With respect to
193
the solubilisation process of water hyacinth an appropriate selection of bacterial strain/
194
culture is necessary. Therefore to determine the ideal isolated bacterial culture providing the
195
highest solubilisation of water hyacinth, sCOD and the VFA of the samples were analysed at 8
ACCEPTED MANUSCRIPT 196
various dosage and time. Fig 1a, 1b, 2a, 2b, 3a and 3b depicts similar trends of increase in
197
sCOD and VFA concentration. sCOD and VFA increased hand in hand as the number of days
198
increased and after reaching the utmost peak, it starts decreasing. The increase in VFA led to
199
the increase in sCOD. For water hyacinth, pretreatment with SFa2 illustrated the highest
200
sCOD (solubilisation) in 4 days followed by BRb2 and UN3d2 (6 days) at a dosage of 109
201
CFU/mL. Although the same dose of inoculum showed the highest solubilisation for the
202
entire three different bacterial strains still the degree of solubilisation and the time required
203
for solubilisation differed. SFa2 illustrated the highest solubilisation of 1.33 times or 33.33%
204
followed by BRb2 (1.26 times or 26.75%) and UN3d2 (1.21 times or 20.94%). The release of
205
extracellular and intracellular biopolymers into the soluble phase is a fundamental factor to
206
assess the proficiency of the pretreatment (Mahdy et al., 2016). Initially, the sCOD and VFA
207
for water hyacinth inoculated with 109 CFU/mL of SFa2 was observed to be 2496 mg/L and
208
764 mg/L respectively. As the time required for solubilising is one of the key administering
209
feature in bacterial pretreatment (Kavitha et al., 2017), the release of soluble organic matter
210
amplifies with an augment in pretreatment time. On the 2nd day of pretreatment, sCOD and
211
VFA enhanced to 2506 mg/L and 1000 mg/L, indicating that the rise in sCOD and VFA may
212
be attributable to exopolymeric matrix discharge rather than the availability of the
213
intracellular polymers. More increment in the pretreatment time to 4 days displayed a radical
214
increase in sCOD and VFA to 4160 mg/L and 1480 mg/L respectively. This radical increase
215
in sCOD designates the secretion of cellulase enzyme by the SFa2 bacteria. Firstly, because
216
the generated compounds are the substrates, as a result the recalcitrant lignocellulosic cell
217
wall complex ruptured resulting in the discharge of extracellular and intracellular polymers
218
into the soluble phase. From the 6th day, both VFA and sCOD started declining. Yuan et al.
219
(2014) demonstrated rapid increase in the concentration of VFA and sCOD during 4 days of
220
microbial pretreatment of corn stalk. High sCOD indicates that huge amount of soluble
9
ACCEPTED MANUSCRIPT 221
organic matter are present for anaerobic digestion and the formed VFA performs a significant
222
responsibility in accelerating the hydrolysis of lignocellulosic material (Yu et al., 2004). The
223
mode of action of the microbial pretreatment can be hypothesised by two credible
224
mechanisms. First of all, the bacteria might have attacked the sturdy outer layer by
225
dissolving/ softening down the lignin resilience thereby increasing the permeability of the
226
water hyacinth. Followed by the second mechanism of the bacterial attack in the inner layer
227
of the water hyacinth and finally leading to the hydrolysis of the cellulose and hemicellulose.
228
The bacteria initially starts consuming the easily available soluble organic matter; once the
229
soluble organic matter is over, the bacteria starts secreting exoenzymes to solubilise the
230
particulate organic matter thereby increasing the quantity of soluble organic matter and the
231
bacterial population, leading to the increase in sCOD. After few days, as the availability of
232
food decreases, the bacteria starts consuming each other, leading to the decrease in sCOD.
233
The outcome designates that the conversion of complex organic matter to soluble organic
234
matter can be time consuming, and the optimum pretreatment time and dosage must be
235
equivalent to that, during which sCOD and the VFA accomplishes the highest peak value.
236
The increased level of VFA and sCOD are a proof of the high hydrolytic and cellulolytic
237
activity of the isolated bacteria especially SFa2. Thus the optimal pretreatment dosage and
238
time is 109 CFU/mL of SFa2 in 4 days for water hyacinth. This optimal pretreatment dosage
239
and time makes sure that the exhaustion of soluble organic matter during microbial
240
pretreatment is reduced and that the soluble organic matter is available for biogas generation.
241
3.2 Effect of microbial pretreatment on the compositional analysis of water hyacinth
242
The main objective of the microbial pretreatment is to rupture the lignocellulosic complex
243
of water hyacinth so that a higher amount of easily degradable organic carbon fraction is bio-
244
accessible. Improved biodegradability of water hyacinth can lead to increased biogas
245
production in a short duration as the recalcitrant lignin softens making it easier for the
10
ACCEPTED MANUSCRIPT 246
microorganisms to feed on the depolymerised cellulose. Therefore compositional analysis
247
was carried out to study the modification in the lignocellulosic composition of the water
248
hyacinth before and after microbial pretreatment (Table 2).
249
Lignin is a complex, aromatic heteropolymer in which its main structural constituents,
250
phenylpropanoid aryl-C3 units, are connected by a diverse range of C-O and C-C
251
bonds. Cellulose is a polysaccharide, composed of a linear string of D-glucose molecules
252
connected by β-(1, 4)-glycosidic bonds. Hemicellulose is an easily hydrolysable branched
253
heterogeneous polymer of pentose (xylose, arabinose), hexose (mannose, glucose and
254
galactose) and acetylated sugars. Enhanced delignification in the pretreated substrate when
255
compared to the untreated was evident as the percentage of soluble lignin increased. SFa2
256
pretreated sample substantiates the utmost increase in the percentage of soluble lignin
257
(3.23±0.8 %) followed by BRb2 (2.93±0.1 %) and UN3d2 (2.82±0.4 %) pretreated when
258
compared to the untreated substrate (2.77±0.3 %). The presence of more soluble lignin
259
signifies the softening of the sturdy lignin. The softened lignin makes the cellulose easily bio-
260
accessible. Repolymerization reaction of the lignin is the main reason behind the slight
261
increase in the percentage of acid insoluble lignin in the pretreated samples (Li et al., 2007).
262
Reduction in the composition of cellulose in pretreated water hyacinth than the untreated is
263
advantageous as it represents enhanced solubilisation. The long string cellulose must have
264
split at some indefinite point by the microorganisms during pretreatment leading to the
265
decline in cellulose (Barua and Kalamdhad, 2017). In this manner the percentage of short
266
string glucose fragments (hemicellulose) in the microbial pretreated water hyacinth increased.
267
Highest reduction in cellulose was observed in SFa2 (30.89±0.2 %) followed by BRb2
268
(32.63±0.3 %) and UN3d2 (33.87±0.8 %) respectively. Conversely, SFa2 pretreated water
269
hyacinth illustrated the increase in the availability of hemicellulose (43.06±0.7 %) than the
270
untreated water hyacinth (27.7±0.2 %). Microbial pretreatment helped in the modification of
11
ACCEPTED MANUSCRIPT 271
the chemical composition and physical configuration of the lignocellulosic water hyacinth.
272
These chemical and physical modifications team up to improve the biodegradability of the
273
water hyacinth and enhance biogas generation. Thus the compositional analysis indicates the
274
availability of the utmost amount of readily degradable soluble organics in the SFa2
275
pretreated water hyacinth.
276
Table 2: Compositional changes in water hyacinth due to microbial pretreatment Microbial
Acid
soluble Acid
insoluble Cellulose (%)
Hemicellulose
consortium
lignin (%)
lignin (%)
--
2.77±0.3
7.93±0.5
36.84±0.8
27.7±0.2
SFa2
3.23±0.8
8.34±0.3
30.89±0.2
43.06±0.7
BRb2
2.93±0.1
8.92±0.6
32.63±0.3
36.02±0.8
UN3d2
2.82±0.4
9.05±0.9
33.87±0.8
30.62±0.9
(%)
277 278
3.3 Molecular identification
279
All the three bacterial isolates i.e., SFa2, BRb2 and UN3d2 were identified
280
phylogenetically (Table 3). 16S rRNA analysis helped in the identification of the naturally
281
isolated three bacterial strains. 16S rRNA analysis technique assists in recognising poorly
282
described or rarely isolated strains thereby leading to the recognition of the isolated novel
283
bacterial strains. 16S rRNA gene sequences analysis is also useful for determining the extent
284
of phylogenetic diversity within diverse groups of bacteria. The phylogenetic tree or
285
dendogram represents the evolutionary relationships between various biological organisms.
286
The phylogenetic analysis (fig 4a) revealed SFa2 to be of the phylum Proteobacteria, which is
287
a major phylum of gram negative bacteria. The outer membrane of Proteobacteria is
288
composed of lipopolysaccharides. The identified Proteobacteria was indicated as Citrobacter
289
werkmanii. The nucleotide sequence of the bacterial strain SFa2 displayed 91 % sequence 12
ACCEPTED MANUSCRIPT 290
identity with their closest relatives. Accession number MF099900 was provided by GenBank
291
for SFa2. UN3d2 (fig 4c) also belonged to the phylum Proteobacteria and was identified as
292
Bordetella muralis with an accession number MF098756. While BRb2 (fig 4b) was a
293
member belonging to the phylum Firmicutes. BRb2 was identified as Paenibacillus sp with
294
an accession number MF187530. An accession number is a unique identifier provided to a
295
DNA or protein sequence record for identifying dissimilar versions of that sequence record
296
and the related sequence over time in a solo information repository. Citrobacter werkmanii
297
(MF099900) pretreatment illustrated the highest solubilisation of water hyacinth. Thereby
298
indicating that Citrobacter werkmanii VKVVG4 will be most beneficial in accelerating the
299
hydrolysis of water hyacinth and enhancing the generation of biogas when compared to the
300
other bacterial isolates. Table 3: Phylogenetic identification of the isolates.
301 302 303
Code
Closest relative
Accession
% Solubilisation
number SFa2
Citrobacter werkmanii
MF099900
33.3
MF187530
26.75
MF098756
20.94
VKVVG4
BRb2
Paenibacillus sp VKVVG1
UN3d2
Bordetella
muralis
VKVVG5
304
3.4 Effect of microbial pretreatment on biogas production
305
BMP test was performed between untreated and microbial (SFa2) pretreated water
306
hyacinth as SFa2 illustrated 1.33 times increase in solubilisation than the untreated water 13
ACCEPTED MANUSCRIPT 307
hyacinth. Freshly pulverised water hyacinth whole plant was inoculated with 109 CFU/mL of
308
SFa2 and was incubated at 37◦C, 150 rpm for 4 days. After 4 days of pretreatment, cow dung
309
was added to the pretreated substrate and employed for BMP study. MSM was provided to
310
supply the necessary macro and micro nutrients to the microorganisms.
311
During the initial week of the anaerobic digestion process, lag phase was observed for
312
both the untreated and microbial (SFa2) pretreated water hyacinth due to the acclimatisation
313
phase of the cow dung with the substrate environment inside the anaerobic reactor (fig 5).
314
After a week of anaerobic digestion, biogas production begins to increase with the increase in
315
anaerobic digestion period. A sharp increase in the cumulative biogas production curve for
316
the SFa2 pretreated water hyacinth was observed. Hence, indicating that SFa2 pretreatment
317
was competent enough to hydrolyse some amount of the complex organic macromolecules
318
available in the water hyacinth by forming more readily biodegradable substances available
319
for anaerobic digestion. Due to augmentation of biogas production rate, the cumulative
320
biogas production amplified incessantly during the digestion period. Significant increase in
321
biogas production was observed during the log phase on account of the rapid multiplication
322
and speedy consumption of the water hyacinth by the methanogens. Further increase in
323
biogas production persisted throughout the log and steady phase. SFa2 pretreated water
324
hyacinth achieved a cumulative biogas production of 2123±10 mL on the 20th day whereas
325
the untreated water hyacinth achieved 1023±17 mL of cumulative biogas on the same day.
326
By the end of 50 days, SFa2 pretreated water hyacinth illustrated a cumulative biogas
327
production of 3737±21 mL whereas untreated water hyacinth illustrated a cumulative biogas
328
production of 3038±13 mL. The increased biogas production from water hyacinth after
329
microbial pretreatment signifies the presence of easily digestible soluble organic matter. The
330
enhanced solubilisation of water hyacinth after microbial pretreatment led to an increase in
331
biogas production. Cordoba et al., 2016 reported a cumulative biogas production of
14
ACCEPTED MANUSCRIPT 332
3960 ± 431 mL in 105 days from spent saw dust after microbial (Gymnopilus pampeanus)
333
pretreatment. There are many previous literatures which states of witnessing the increase in
334
biogas production after biological pretreatment of corn straw and saw dust waste respectively
335
(Zhong et al., 2011; Ali et al., 2017). Hydrolysis of untreated water hyacinth is a slow process
336
and yields less biogas when compared to the SFa2 pretreated water hyacinth. Microbial
337
pretreatment immensely influences the rate of the anaerobic digestion process and
338
biodegradability of the water hyacinth. This highlights the fact that the discharge of soluble
339
organic matter during microbial pretreatment helps in the biodegradability of the substrate,
340
enhancing biogas yield and process kinetics. Microbial (SFa2) pretreatment provoked desired
341
structural and chemical modification in the lignocellulosic water hyacinth, thereby enhancing
342
the biomethanation process. As, the lignocellulosic complex of the untreated water hyacinth
343
was tightly intact, slow hydrolysis period was witnessed. In other words the presence of
344
lignin barrier and crystalline long cellulosic fibres in the untreated water hyacinth hindered
345
the microorganisms to work synergistically, ultimately producing lesser quantity of biogas.
346
Sturdy lignin and crystalline cellulose requires more time to disintegrate into labile carbon
347
moieties. Thus, microbial pretreatment was adequately efficient for rapid biodegradation of
348
the water hyacinth leading to faster hydrolysis and enhanced biogas production.
349
3.5 Scaled up batch anaerobic digestion and its kinetics
350
As all the above analytical studies and biogas production studies demonstrated positive
351
results therefore the batch study was scaled up to 20 L in order to check the operational
352
conditions and variations to be encountered during scaled up process.
353
From Fig 7, the massive increase in biogas production from water hyacinth after
354
microbial pretreatment is convincingly evident. Cumulative biogas production from water
355
hyacinth after microbial pretreatment was observed to be 102491±13 mL in 80 days whereas
356
a cumulative biogas production of 50612±18 mL only was obtained from untreated water 15
ACCEPTED MANUSCRIPT 357
hyacinth for the same number of days. An increase of 3.07 times in biogas production was
358
observed in the 20 L batch study. The highest daily biogas production of 4898±10 mL was
359
achieved on 21st day after microbial pretreatment of water hyacinth. Attaining the highest
360
daily biogas production on 21st day was quite faster than the untreated water hyacinth. The
361
highest daily biogas production of untreated water hyacinth was achieved on the 28th day.
362
This indicates the acceleration in the hydrolysis phase of water hyacinth after microbial
363
pretreatment. Even the amount of highest daily biogas production from the untreated water
364
hyacinth (2016±13 mL) on the 28th day was far lesser when compared to the highest daily
365
biogas production of 4898±10 mL achieved on 21st day after microbial pretreatment of water
366
hyacinth. A dynamic change in the generation of biogas was witnessed in the water hyacinth
367
after microbial pretreatment. The enhanced amount of biogas production from water hyacinth
368
after microbial pretreatment denotes the presence of excessive amount of easily soluble
369
compounds. In the untreated water hyacinth due to the presence of recalcitrant lignin and
370
crystalline cellulose, the activity of the microorganisms was slow. The effective strain and
371
ideal culture condition made the pretreatment process efficient by reducing the hydrolysis
372
period almost by a week and by enhancing biogas production.
373
The acquired estimated kinetic values are provided in table 4. The values signifies that
374
the modified Gompertz equation can be employed to estimate the maximum methane
375
generation potential, the maximum methane generation rate and the lag phase. The kinetic
376
parameters of the untreated and microbial pretreated water hyacinth utilised during 20 L
377
batch study was determined where the values of M of the pretreated water hyacinth (171.039
378
L CH4) was proved to be better than the untreated water hyacinth (71. 796 L CH4). R2 value
379
surpassed 0.90 for both untreated and microbial pretreated water hyacinth, suggesting that
380
methane generation can be well simulated. Thus, the curve of the modified Gompertz
381
equation fitted well with the experimental values of cumulative biogas generation (Fig 6). 16
ACCEPTED MANUSCRIPT 382
Table 4: Kinetics values of untreated and microbial pretreated water hyacinth used in 20 L
383
batch study. Substrate
M
Rmax
λ (d)
(L CH4)
(L CH4 d-
R2
Y (L CH4)
1)
Pretreated
171.039
0.1000
0.0000
0.96
102.49
Untreated
71. 796
0.0772
0.0010
0.92
50.65
384 385
386
4. CONCLUSION Microbial pretreatment of water hyacinth proved to be proficient in accelerating the
387
hydrolysis step of anaerobic digestion by improving the biodegradability of the substrate.
388
Pretreatment of water hyacinth with SFa2 (Citrobacter werkmanii VKVVG4, MF099900)
389
helped in achieving the highest solubilisation of 33.33% with an optimum dosage of 109
390
CFU/mL within 4 days. Both BMP (1 L) and batch (20 L) assay revealed that the cumulative
391
methane production of SFa2 pretreated water hyacinth was higher than the untreated water
392
hyacinth. 1 L BMP assay illustrated that SFa2 pretreated water hyacinth attained a cumulative
393
biogas production of 3737±21 mL whereas untreated water hyacinth attained a cumulative
394
biogas production of 3038±13 mL on the 50th day. Thus, Citrobacter werkmanii VKVVG4 or
395
SFa2 pretreatment of water hyacinth succeeded in enhancing biogas production also.
396
Acknowledgement
397 398
The authors are grateful to Mr. Vikas Kumar, Centre for Energy, Indian Institute of Technology Guwahati, Assam, India.
399 17
ACCEPTED MANUSCRIPT 400
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ACCEPTED MANUSCRIPT (a) 5000 sCOD (mg/L)
4500 4000 3500 3000 2500 2000 1500 1000
Control
500
10*8 0
2
4
6 Time (Days)
8
10
10*9 10*10
(b) 1500 1400 VFA (mg/L)
1300 1200 1100 1000 900 800 700 600 500 0
2
4
6 (Days)8 Time
10
12
Fig 1: Changes in (a) sCOD and (b) VFA concentration during microbial pretreatment with SFa2.
ACCEPTED MANUSCRIPT (a) 4500 4000 sCOD (mg/L)
3500 3000 2500 2000 1500 1000
Control 10*8
500 0
2
4
6 Time (Days)
8
10
12
10*9 10*10
(b) 4000
VFA (mg/L)
3500 3000 2500 2000 1500 1000 500 0
2
4
6 8 Time (Days)
10
12
Fig 2: Changes in (a) sCOD and (b) VFA concentration during microbial pretreatment with BRb2.
ACCEPTED MANUSCRIPT (a) 4000
sCOD (mg/L)
3500 3000 2500 2000 1500 1000
Control
500 0
2
4
6
8
10
10*8
12
10*9
Time (Days)
10*10
(b) 1200
VFA (mg/L)
1100 1000 900 800 700 600 500 0
2
4
Time (Days) 6 8
10
12
Fig 3: Changes in (a) sCOD and (b) VFA concentration during microbial pretreatment with UN3d2
ACCEPTED MANUSCRIPT Cumulative biogas produced (mL)
4500 untreated
4000
pretreated
3500 3000 2500 2000 1500 1000 500 0 0
5
10Time15(Days) 20
25
30
35
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45
50
Fig 4: Cumulative biogas production of untreated and pretreated water hyacinth. (a)
(b)
(c)
Fig 5: Phylogenetic tree based on 16S rRNA gene sequences of bacterial strain (a) SFa2 (Citrobacter werkmanii VKVVG4) (b) BrB2 (Paenibacillus sp. VKVVG1) and (c) Un3d2 (Bordetella muralis VKVVG5)
(a)
Cumalative biogas production (mL)
ACCEPTED MANUSCRIPT
70000 60000 50000 40000 30000
Measured Biogas production
20000
Predicted biogas production
10000 0 0
10
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Time (Days)
Cumulative Biogas Production (mL)
(b) 120000 100000 80000 60000
Measured Biogas Production
40000
Predicted Biogas Production
20000 0 0
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Time (Days)
Fig 6: Cumulative biogas production experimental data fitted with Gompertz predicted data for (a) untreated water hyacinth and (b) pretreated water hyacinth.
ACCEPTED MANUSCRIPT
Cumulative Biogas Produced (mL)
160000
Untreated
140000
Pretreated
120000 100000 80000 60000 40000 20000 0 0
10
20
Time 30 (Days) 40
50
60
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80
Fig 7: Cumulative biogas production of untreated and pretreated water hyacinth in 20 L anaerobic batch digester in a time period of 80 days.
ACCEPTED MANUSCRIPT HIGHLIGHTS
Novel bacterial pure cultures were utilized to pretreat water hyacinth. Microbial pretreatment showed enhanced solubilisation of water hyacinth. Citrobacter werkmanii VKVVG4 illustrated highest solubilisation of water hyacinth. Microbial pretreatment improved biogas production from water hyacinth.