Acidogenic spent wash valorization through polyhydroxyalkanoate

(PHA) synthesis coupled with fermentative biohydrogen production

K. Amulya, M. Venkateswar Reddy, S. Venkata Mohan

Bioengineering and Environmental Centre (BEEC), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India
h i g h l i g h t s
PHA production from B. tequilensis and SWE was evaluated for the rst time.
Produced co-polymer P (3HB-co-3HV) showed higher fraction of HB.
Integration of PHA and H
2
production reduces overall production and treatment cost.
a r t i c l e i n f o
Article history:
Received 24 December 2013
Received in revised form 3 February 2014
Accepted 8 February 2014
Available online 17 February 2014
Keywords:
Spent wash efuents
B. tequilensis
Acidication
Volatile fatty acids (VFA)
Waste valorization
a b s t r a c t
The production of polyhydroxyalkanoates (PHAs) by Bacillus tequilensis biocatalyst using spent wash
efuents as substrate was evaluated to increase the versatility of the existing PHA production process
and reduce production cost. In this study, spent wash was used as a substrate for biohydrogen (H
2
) pro-
duction and the resulting acidogenic efuents were subsequently employed as substrate for PHA produc-
tion. Maximum H
2
production of 39.8 L and maximum PHA accumulation of 40% dry cell weight was
attained. Good substrate removal associated with decrement in acidication (53% to 15%) indicates that
the VFA generated were effectively utilized for PHA production. The PHA composition showed presence
of copolymer [P (3HB-co-3HV)] with varying contents of hydroxybutyrate and hydroxyvalerate. The
results obtained suggest that the use of spent wash efuents as substrate can considerably reduce the
production cost of PHA with simultaneous waste valorization. PHA synthesis with B. tequilensis and spent
wash efuents is reported for the rst time.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
Hydrogen is regarded as a sustainable and viable alternative
energy source whose consumption does not lead to any CO
2
emissions. With respect to sustainable development and waste
minimization issues, bio-hydrogen (H
2
) production from renew-
able sources has received considerable attention in recent years
(Kim et al., 2012; Venkata Mohan et al., 2013). Production of clean
energy with simultaneous valorization of waste materials makes
biological hydrogen production process a promising approach for
meeting the mounting energy needs of the world. Extensive re-
search has been carried out on H
2
production using different types
of wastewaters like food waste, municipal waste, distillery efu-
ents etc. Venkata Mohan et al. (2008) and Searmsirimongkol
et al. (2011) have reported H
2
production from distillery spent
wash with concurrent wastewater treatment. H
2
production was
reported to improve when a co-culture (Citrobacter freundii 01,
Enterobacter aerogenes E10 and Rhodopseudomonas palustris) was
used with sugar cane distillery efuent as substrate (Vatsala
et al., 2008). However, one of the major drawbacks of using organic
waste as a substrate is lower H
2
yield and existence of about 60
70% of the original organic matter as residue in the wastewater
(Venkata Mohan et al., 2010, 2013). In addition, large amounts of
soluble acid metabolites e.g. volatile fatty acids (VFA) are gener-
ated that mandate further treatment to evade the negative envi-
ronmental impacts associated with their disposal. VFAs are the
precursors of polyhydroxyalkanoates (PHA) in microbial metabo-
lism. Production of PHAs under dened microaerophilic conditions
using the VFA rich efuents from the biohydrogen producing reac-
tor, as main substrate is an alternate and highly sustainable valori-
zation technique (Venkata Mohan et al., 2010; Passanha et al.,
2013; Reddy et al., 2013).
In an increasingly globalized world, plastics have turned out
to be one of the most widely used materials in all facets of life
such as packaging, home appliances, computer equipment, bottles,
http://dx.doi.org/10.1016/j.biortech.2014.02.026
0960-8524/ 2014 Elsevier Ltd. All rights reserved.

Corresponding author. Tel./fax: +91 40 27191664.

E-mail address: vmohan_s@yahoo.com (S. Venkata Mohan).
Bioresource Technology 158 (2014) 336342
Contents lists available at ScienceDirect
Bioresource Technology
j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
disposable items and automobiles (Jin et al., 2013; Lee and Gil-
more, 2005). The usage of these non-biodegradable plastics will
have potentially damaging ramications on the environment and
hence considerable emphasis has to be laid on the development
of biopolymer materials like PHA that are eco-friendly (Akaraonye
et al., 2010) and biodegradable (Jin et al., 2013). PHAs are
linear aliphatic polyesters that accumulate as cytoplasmic
inclusions in various bacteria and archaea under certain nutrient
deprived growth conditions such as excess supply of carbon
and lack of one or more essential nutrients, e.g. oxygen, nitrogen,
phosphorus, sulfur and trace elements such as magnesium,
calcium, and iron (Hazer and Steinbuchel, 2007). Poly-b-hydroxy-
butyrate (PHB) and poly-b-hydroxyvalerate (PHV) are the most
common forms of PHA accumulated by microorganisms (Luengo
et al., 2003).
Over the past few years, research has been focused on the use of
synthetic fatty acids and low cost agro-industrial surplus feed
stocks (Albuquerque et al., 2011) and fermented paper mill efu-
ents (Bengtsson et al., 2008) as prime substrates for PHA produc-
tion. The integration of PHA production with simultaneous
valorization of acidogenic efuents is a fairly recent innovation
(Ntaikou et al., 2009; Passanha et al., 2013). Santimano et al.
(2009) have reported use of cane molasses as substrate for the
growth of Bacillus sp COLI/A6 (biomass content 6 g l
1
dry cell
weight (DCW)) and subsequent PHA production (54.68% DCW).
Omar and co-workers also achieved 50% (DCW) of P(3HB) using
Bacillus megaterium grown on beet molasses (Omar et al., 2001).
PHA production using synthetic acids and food waste, by Bacillus
tequilensis has also been reported (Reddy et al., 2013). In view of
the emerging interest in the use of fermented substrates as feed-
stocks for PHA production this work has been aimed at evaluating
the possibility of using VFA rich spent wash efuents from biohy-
drogen production process as substrate and B. tequilensis as the
biocatalyst for PHA production. This study also helps in under-
standing the inuence of different types of VFAs on polymer com-
position. This integration approach can dramatically reduce
production costs with simultaneous waste valorization, for a clea-
ner and more sustainable society. There are no reports in literature
till date that describe the production of PHA with B. tequilensis
using spent wash efuents (SWE) as substrate.
2. Methods
2.1. Biocatalyst
Acidogenic H
2
production experiments were carried out using
anaerobic consortium as inoculum collected from a full scale
anaerobic reactor treating composite wastewater. Prior to inocula-
tion, the parent culture was washed in phosphate saline buffer
(5000 rpm, 20 C) and enriched with designed synthetic wastewa-
ter (DSW) [glucose 3 g/l; NH
4
Cl 0.5 g/l, KH
2
PO
4
0.25 g/l,
K
2
HPO
4
0.25 g/l, MgCl
2
0.3 g/l, CoCl
2
25 mg/l, ZnCl
2

11.5 mg/l, CuCl
2
10.5 mg/l, CaCl
2
5 mg/l, MnCl
2
15 mg/l, FeCl
3
25 mg/l, NiSO
4
16 mg/l] under anaerobic microenvironment at
pH 6.0 in conical ask under shaking (80 rpm; 48 h). B. tequilensis
was used as biocatalyst for PHA production studies and the
organism was isolated from PHA producing bioreactor by direct
enrichment techniques (Reddy and Venkata Mohan, 2012a).
The genomic DNA was extracted and puried from the isolated
pure colonies using phenolchloroform method. The variable V3
region of 16S rDNA was amplied by PCR using primers 8F
(5
0
-AGAGTTTGATCCTGGCTCAG-3
0
) and 1542R (5
0
-AAGGAGGT-
GATCCAGCCGCA-3
0
). The nucleotide sequence was deposited in
the GenBank database under accession number HE612876
(Reddy and Venkata Mohan, 2012a). Stock culture was preserved
on nutrient agar slants overlaid with 20% (v/v) glycerol and kept
at 20 C.
2.2. Wastewater
Distillery spent wash (pH, 4.2; VFA, 27 g/l; chemical oxygen
demand (COD), 126 g/l; Total dissolved solids (TDS), 112 g/l;
and Total solids (TS), 113 g/l) was used as substrate for acido-
genic H
2
production. The outlet of H
2
producing reactor viz.,
spent wash efuents (SWE) (pH, 3.83; VFA, 12 g/l; COD,
16.80 g/l; carbohydrates, 13.75 g/l) was used as substrate for
PHA production. Prior to loading, the efuent was adjusted to re-
quired organic loading rates (OLR1, 0.66; OLR2, 1.32; OLR3, 1.98;
OLR4, 2.64 kg COD/m
3
-day) with tap water. After adjusting the
organic load the SWE was autoclaved and used as substrate for
PHA production.
2.3. Experimental methodology
2.3.1. Biohydrogen
Anaerobic bioreactor (working volume, 18 L; gas holding capac-
ity, 4.51 L) operated in periodic-discontinuous batch mode with a
total cycle period of 72 h was used to produce biohydrogen. The
bioreactor was operated at ambient temperature (29 2 C) under
anaerobic microenvironment with spent wash by adjusting the
COD load to 30 g/l and pH to 6.0. The efuents (rich in VFA,
SWE) collected from the bioreactor after 48 h were used as pri-
mary-substrate for PHA production.
2.3.2. Bioplastics
The feasibility of bioplastics production in the form of PHA was
evaluated using B. tequilensis as biocatalyst in presence of SWE as
substrate. The pH was adjusted to 7.0 0.1 using 1 N NaOH. After
adjusting the organic load and pH, the wastewater was sterilized
by autoclaving (121 C; 15 lb; 15 min) and then transferred to
reactors with a total/working volume of 250/100 ml. Prior to start-
up, the reactors containing 100 ml of substrate were inoculated
with 4% of overnight grown B. tequilensis. The reactors were sub-
jected to continuous mixing (120 rpm) and were maintained at
ambient room temperature (29 2 C). In order maintain aerobic
microenvironment and to ensure the exchange of gases, the reac-
tors were closed with cotton plugs.
2.4. Extraction and quantication of bioplastics
Extraction and estimation of PHA was performed following the
procedure reported elsewhere (Law and Slepecky, 1960; Reddy
et al., 2012b). The biomass pellet was collected and washed with
acetone and ethanol separately to remove unwanted materials.
The pellet was suspended in 4% sodium hypochlorite and incu-
bated at room temperature for 3 h. The resulting mixture was cen-
trifuged (3000g for 30 min at 10 C) and the pellet (with lysed
cells) was again washed with acetone and ethanol prior to dissolv-
ing in hot chloroform. To separate the polymer from cell debris the
pellet was passed through glass ber lter (0.45 lm pore size). The
chloroform ltrate thus obtained was used to estimate PHA color-
imetrically. The chloroform from the ltrate was evaporated and
10 ml of sulphuric acid (36 N) was added, which converts the poly-
mer to crotonic acid. The resultant solution was heated at 100 C
on a water bath for 10 min. The solution was cooled and its absor-
bance was measured at 235 and 285 nm for determining the hy-
droxyl butyrate (HB) and hydroxyvalerate (HV) concentration
respectively, against a sulfuric acid blank. Standard curve was pre-
pared using pure poly (3hydroxybutyrate-co-3hydroxyvalerate), P
(3HB-co-3HV) (co-polymer; natural origin, Aldrich). FTIR spectro-
scopic analysis was also performed to conrm the presence of PHA.
K. Amulya et al. / Bioresource Technology 158 (2014) 336342 337
2.5. Analysis
Biohydrogen generated during the fermentation was estimated
using a microprocessor based pre-calibrated H
2
sensor (electro-
chemical 3 electrode H
2
sensor, FMK satellite 420 mA version,
ATMI GmbH Inc., Germany) (Venkata Mohan et al., 2008). The bio-
gas was collected by water displacement method through an outlet
provided at the top of the reactor and was quantied using electro-
chemical sensor/gas chromatography. Bioprocess was monitored
based on pH, VFA, and COD (closed reuxing method) estimations
according to standard methods (APHA, 1998). Quantitative estima-
tion of VFA was carried out by high performance liquid chromatog-
raphy (HPLC; Shimadzu LC10A). Dehydrogenase enzyme activity of
B. tequilensis was estimated by the method described previously
(Reddy et al., 2010; Zhenhua et al., 2008). To determine the decom-
position temperature of PHA, the samples were subjected to ther-
mo gravimetric analysis (TGA) (Mettler Toledo). 5 mg of the
sample was heated in an aluminum pan at a temperature range
of 50900 C under nitrogen atmosphere, at a heating rate of
5 C. Electron discharge pattern of B. tequilensis during PHA pro-
duction was evaluated by employing cyclic voltammetry (CV)
using potentiostatglavanostat system (Ecochemie) by applying a
potential ramp at a scan rate of 30 mV/s over the range of +0.5 to
0.5 V to the working electrode. An electrochemical cell having
platinum and graphite as working and counter electrodes respec-
tively, against AgAgCl (S) reference electrode was used for carry-
ing out the analysis. All the electrochemical assays were performed
with B. tequilensis in whole cell form by using SWE as electrolyte.
3. Results and discussion
3.1. Biohydrogen production
H
2
production started from 2nd h of cycle operation and in-
creased up to 8th h and then showed decrement during the stabi-
lized phase of operation. Cumulative H
2
production of 39.8 L was
observed with specic H
2
yield of 142 l/kg COD
R
. The biogas mainly
comprised of H
2
(21%), CH
4
(36%) and CO
2
(43%). Substrate degra-
dation efciency increased with time and reached a maximum of
44% at the end of the cycle during stabilized phase of operation.
The resulting efuent from bioreactor was highly acidic (pH,
4.8 0.2) in nature due to the presence of high concentration of
VFAs (12 0.21 g/l) formed as co-metabolites along with H
2
.
Acidication of waste due to VFA formation can be calculated
using the relation (Alkaya and Demirer, 2011).
Acidification % VFA
t
COD
t
=100 1
where; VFA
t
and COD
t
represent total VFA concentration (mg/l) and
COD concentration (mg/l) at a particular time interval, respectively.
During the H
2
production process, 2 days of HRT yielded higher VFA
concentrations (12 0.21 g/l), which indicates high acidogenic
activity. At the end of cycle operation, the acidication was
48.93%, which was quite high. The main acidication products were
acetic acid (4.87 0.2 g/l), butyric acid (2.96 0.4 g/l), propionic
acid (2.86 0.4 g/l) and valeric acid (0.84 0.4 g/l). Substrate char-
acteristics and operational conditions play a major role on product
composition in an acidication reactor (Alkaya and Demirer, 2011).
Production of high amounts of acetic, butyric and propionic acids
can be associated with substrate degradation and also correlates
with previous studies in which short-chain fatty acids were found
to be dominant in acidogenic reactors (Yu and Fang, 2002; Dinopou-
lou et al., 1988). The acidied efuent generated from H
2
reactor
was subsequently used as a primary-substrate for bioplastic
production after adjusting the organic load using tap water and
pH using 1 N HCl or 1 N NaOH.
3.2. Bioplastics production
3.2.1. Biomass growth
B. tequilensis is a Gram-positive, motile, rod shaped aerobic
organism. There are no literature reports till date regarding the
production of PHA with B. tequilensis using spent wash efuents
(SWE) as substrate. This organism has phosphorous solubilization
activity, capacity to produce lipase enzyme (150 U/ml) under opti-
mized conditions (Chakravarthy and Narasu, 2012) and capability
to produce an extracellular thermo alkaliphilic protease (Khan
et al., 2011). The growth of B. tequilensis during batch culture under
aerobic conditions based on dry cell weight (DCW) is depicted in
Fig. 1(a). In all the OLRs studied, a marked improvement in DCW
was observed between 0 and 6 h followed by stabilization up to
36 h. Since overnight grown culture was used for the experiment
it can be considered that the initial physiological adaptation period
was eliminated and a drastic growth was observed. Thus, the rep-
resentative growth phases can be dened as the exponential phase
at 6 h followed by stationary phase at 36 h. The maximum DCW of
7.8 g/l was observed at 36th h for OLR2 operation followed by
4.8 g/l in OLR3 (12th h), 4.6 g/l in OLR1 (36th h) and 3.9 g/l in
OLR4 (12th h).
3.2.2. PHA
Figure 1(b) illustrates the PHA production capacity of B. tequil-
ensis with SWE. Signicant variations in PHA production were visu-
alized with the function of time and organic load. Maximum PHA
production (1740%) was observed during the 36th h of operation
in all the OLRs expect for OLR4, which showed maximum PHA pro-
duction (8.67%) at 30th h. OLR2 showed highest PHA production of
40% followed by OLR1 (36%) and OLR3 (17%). In all the OLRs stud-
ied a gradual increment in the PHA accumulation was observed
from 0 to 36 h and decreased marginally thereafter. OLR3 and
OLR4 showed lower PHA production due to high substrate concen-
tration that might have caused load shock resulting in suppressed
bacterial growth. The initial acid concentration is crucial for main-
taining the activity of cells. After loading the substrate (VFA) the
acid molecules penetrate into the cells and dissociate in the cyto-
plasm and the tolerance to these dissociated acid molecule by
the cells is vital (Wang and Yu, 2000). At higher OLR, presence of
high acid concentrations might have decreased the cell activity
and reduced the acid utilization rate, thereby lowering the PHA
production. A conspicuous difference was not observed in the
PHA production capacity of the biocatalyst at OLR1 and OLR2, sig-
nifying that these concentrations were ideal for bacterial growth as
well as PHA production. Previous study with B. tequilensis docu-
mented 59% of PHA yield from synthetic acids (SA) and 36% from
acidogenic fermented food waste (AFW) (Reddy et al., 2013). While
in the present study, the yield obtained with aciodgenic SWE is
marginally higher (40%) than that obtained from AFW. This illus-
trates that the complex acidogenic SWE is able to provide ideal
environment and supplement key nutrients that are effectively uti-
lized for PHA synthesis.
3.2.3. Fatty acid Vs PHA
Composition of PHA based on co-polymer, P (3HB-co-3HV) syn-
thesis was studied with OLR2. Overall, the polymer obtained
showed high ratio of hydroxybutyrate (HB) (60%) to hydroxyvaler-
ate (HV) content (40%) (Fig. 1(c)). Using a mixture of acids such as
acetate, propionate, butyrate and valerate facilitates the produc-
tion of a co-polymer of HB and HV (Lemos et al., 2006). According
to the type of VFA fed to the culture, the monomer composition of
the PHA differs and also plays an essential role in varying the
mechanical and thermal properties of PHA. This can be further sup-
ported by previous study by Reddy et al., (2013) where high HB
content (SA, 82%; AFW, 89%) and low HV content (SA, 15%; AFW,
338 K. Amulya et al. / Bioresource Technology 158 (2014) 336342
9%) were obtained, since acetate and butyrate were the major
sources of VFA present that were effectively utilized for PHA pro-
duction. However, SWE documented higher HV content (40%) as
against the HV content obtained in AFW and SA. This difference
in the HV contents might be due the presence, as well as the utili-
zation of both propionic and valeric acids present in the SWE.
Individual VFA composition was analyzed at OLR2 using HPLC
at specic time intervals (Fig. 2(a)). In the 0th h, the SWE majorly
consisted of acetic, butyric, propionic and valeric acids associated
with other acids in very low concentrations. A decrement in VFA
concentration was observed with operation time. Among the four
acids, acetic acid showed highest removal compared to butyric
acid, propionic acid and valeric acid. At the end of the cycle, the
concentration of acetic acid was 268 mg/l, butyric acid was
186 mg/l, propionic acid was 41 mg/l and valeric acid was 41 mg/
l. The higher removal of acetic and butyric acids when compared
to propionic and valeric acid correlates well with the PHA compo-
sition, where HB fraction was relatively higher when compared to
HV. The utilization of acetate and butyrate favors HB production
while the utilization of propionate and valerate favors HV produc-
tion. The presence of high amounts of acetic acid might have fa-
vored the high PHA production in OLR1 (2400 mg/l) and OLR2
(1681 mg/l) operation. Lemos et al. (2006) observed that the high-
est polymer yield was obtained for acetate, followed by butyrate,
propionate and valerate when used as substrates, since propionate
and valerate have to be carboxylated to produce acetyl-CoA which
might be a reason for lower polymer yields. The results obtained
are also in accordance with what has been previously reported
by Albuquerque et al. (2011) where co-polymers of HB and HV
were obtained by manipulating the feeding regimen for PHA
production.
3.2.4. Characterization of PHA
The different conformational bands related to PHA were ana-
lyzed using FTIR (Fig. S1). The absorption band at 1025 cm
1
repre-
sents CAOAC chemical groups of the polymer. The absorption
bands in the region 3435 cm
1
, 2924 cm
1
and 1634 cm
1
corre-
spond to the stretching of CH
3
asymmetric, CH
2
anti-symmetrical
and methyne (ACH) group, respectively. The thermal stability of
the polymers was analyzed by TGA. Temperature at 5% weight loss
was used as the decomposition temperature (T
d
) to evaluate the
thermal stability (Fig. S2). While the decomposition of PHA starts
at 205 C, derivative thermogravimetric (DTG) analysis showed
that the temperature corresponding to maximum degradation rate,
i.e. D
max
was 345 C for PHA. These results suggest the presence of
the co-polymer (3HB-co-3HV).
3.2.5. pH
pH of the feed was adjusted to 7 using 1 N NaOH before feeding
to the reactors as neutral pH is favorable for PHA accumulation
than acidic and high alkaline pH. The enzymes involved in PHA
production are active at neutral pH. Higher extracellular pH in-
duces a negative electrical difference across the cell membrane,
Fig. 1. Variations in (a) DCW against time with the function of organic load; (b) PHA
recovered from Bacillus tequilensis at different OLRs during different time intervals;
(c) composition of PHA recovered from Bacillus tequilensis at OLR2.
Fig. 2. (a) Variation in VFA concentration during PHA production from Bacillus
tequilensis, at OLR 2 and (b) pH prole during various OLRs.
K. Amulya et al. / Bioresource Technology 158 (2014) 336342 339
leading to the higher energy requirement for VFA uptake (Filipe
et al., 2001). OLR1 showed highest increment in pH (78.8), fol-
lowed by OLR2 (78.5), OLR4 (78.4) and OLR3 (78.25)
(Fig. 2(b)). Obtained pH values correlated well with VFA removal
efciency prole.
3.2.6. COD removal
Substrate removal increased as a function of time, signifying the
efciency of the system towards treatment (Fig. 3(a) and (b)). Low-
est OLR showed the highest removal efciency and vice versa. This
phenomenon might be due to the presence of high substrate com-
plexity in OLR4, which might cause inhibition of the bacterial
growth. OLR1 showed highest COD removal efciency (73%), fol-
lowed by OLR2 (64%), OLR3 (59%) and OLR4 (55%). Using Eq. (1),
the % acidication at OLR2 was calculated (Fig. 3(c)). At 0th h the
% acidication was 53%, which showed a gradual decrement by
the end of the cycle (13.8%). This indicates that the VFAs produced
in acidogenic fermentation were effectively utilized for PHA syn-
thesis. This integration strategy is highly feasible for the simulta-
neous treatment of SWE and their valorization as a renewable
resource for PHA production.
3.2.7. Dehydrogenase activity
Biological oxidation of organic compounds is a dehydrogenation
process mediated by many different intracellular including specic
dehydrogenases. The dehydrogenase (DH) enzyme activity appar-
ently reects the activity of biocatalyst (Sun et al., 2012). The activ-
ities of various dehydrogenases, which are a fundamental part of
the enzyme system of all microorganisms serve as an indicator
of the microbiological redox systems and may be considered a
good measure of microbial oxidative activities. DH is the main
enzyme involved in glycolytic pathway and converts glucose to
acetyl-CoA, which is further converted to PHA. Since all anaerobic
and aerobic bacteria possess electron transport systems, tetrazo-
lium salt like triphenyltetrazolium chloride (TTC) is used as an arti-
cial electron acceptor to detect DH activity in metabolically active
bacteria (Griebe et al., 1997). The initial DH activity (0 h, 0.11 lg/
ml) was same for all the OLRs studied. DH activity increased with
time, OLR2 and OLR4 showed maximum enzyme activity (48 h,
1.29 lg/ml) followed by OLR3 (48 h, 0.93 lg/ml), and OLR1 (48 h,
0.91 lg/ml). Marked improvement in the DH activity was recorded
in between 30 and 36 h in all studies, which correlates well with
the high PHA production observed at that time. The DH activity
showed further increment by the end of the experiment, indicating
the active role of the enzyme in substrate metabolism (Fig. 3(d)).
DH enzyme mainly involves in oxidation and reduction reactions
occurring in the cell. Many specic dehydrogenases transfer H
+
to either Nicotinamide adenine dinucleotide (NAD) or Nicotin-
amide adenine dinucleotide phosphate (NADP).
3.2.8. Bio-electrocatalytic activity
The bio-electrocatalytic behavior of the biocatalyst in terms of
electron discharge was studied by employing cyclic voltammetry
(CV), which permits direct electrochemical detection of the redox
signals and senses the potential difference across the interface.
Voltammograms (Vs Ag/AgCl (S)) recorded in situ visualized
marked variation in the electron discharge properties with the
function of nature of biocatalyst. The voltammetric prole of B.
tequilensis represented a pattern similar to the typical growth
curve of bacteria which is characterized by a lag phase followed
by log and decline phases. Initially, lower redox catalytic currents
(oxidation currents (OC): 0.11 lA; reduction currents (RC):
0.32 lA) were recorded which were almost similar till 12 h of oper-
ation. Later, an exponential phase was observed after 12 h which
Fig. 3. (a) COD removal, (b) COD concentration, (c) VFA acidication proles at OLR2 and (d) DH enzyme activity during PHA production from Bacillus tequilensis.
340 K. Amulya et al. / Bioresource Technology 158 (2014) 336342
recorded higher redox catalytic currents at 24 h (OC: 0.27 lA; RC:
0.56 lA) of operation. Comparatively higher RC was observed than
the OC attributing to the formation of end products (PHA) by the
terminal electron acceptor (VFA). Higher reduction indicates the
capability of system in effectively forming the end products
through active neutralization reactions. The exponential phase also
relates to the metabolic activities of the biocatalyst where PHA
production was also higher supporting the active polymer storage
capabilities of the biocatalyst. After the exponential phase, a steady
decline in OC was observed after 24 h till the end of operation (OC:
0.14 lA; RC: 0.20 lA). This represents the decline phase where the
availability of substrate to the biocatalyst was exhausted. Whereas
the RC showed a sharp drop at 36 h (0.17 lA) after which the
behavior was found to be stabilized (0.20 lA). The voltammograms
depicted clear functioning of the metabolic activities of the biocat-
alyst throughout the operation (Fig. 4(a); Table 1).
To understand the electron discharge efciency of the system,
electrochemical and kinetic parameters pertaining to the biocata-
lyst behavior with the function of operating conditions were eval-
uated. Tafel analysis is the electroanalytical technique used for
evaluating the behavior of the biocatalyst based on proton and
electron transfer (Venkata Mohan and Srikanth, 2011). A Tafel plot
was constructed with each time interval to calculate the kinetic
parameters such as oxidative slope (ba), reductive slope (bc) and
polarization resistance (R
p
in O). Lower Tafel slope indicates higher
electrocatalytic activity along with electron transfer efciencies
while higher Tafel slope indicates lower electrocatalytic activity
and electron transfer efciencies. Reductive slopes were lower
than oxidative slopes irrespective of the time indicating the feasi-
bility for higher reduction reactions. However, based on the time
intervals, variations in slopes were observed. The oxidative slopes
were observed to be higher during 0 h (0.591 V/dec) and 48 h
(0.548 V/dec) of operation. Lower oxidative slopes were observed
at 24 h (0.382 V/dec) followed by 36 h (0.482 V/dec). Reductive
slopes also followed the same trend where the highest slope was
observed at 0 h (0.203 V/dec). During the 12 h (0.158 V/dec) a dec-
rement in the slope was observed which again showed an incre-
ment and was almost similar during 24 h (0.166 V/dec) and 36 h
(0.166 V/dec) followed by a drop at 48 h (0.15 V/dec). Among all
the time intervals, the redox Tafel slopes at 24 h were signicantly
low when compared to other time intervals indicating that both
oxidation and reduction reactions were favored. Lower reduction
slopes infer higher reduction reactions featuring the activity of bio-
catalyst towards PHA synthesis. During substrate metabolism, the
protons and electrons that are generated will further get reduced
with VFA in the PHA production pathway, which might have con-
tributed for the higher redox reactions depicting lower redox Tafel
slopes (Fig. 4(b)). Polarization resistance calculated from the Tafel
analysis was relatively low at 24 h (3.494 O), followed by 36 h
(4.773 O), 12 h (4.811 O), 48 h (5.291 O) and 0 h (5.774 O)
(Fig. 4(c)). Low polarization resistance observed at 24 h supported
the transfer of higher number of reducing equivalents from the
biocatalyst towards reduction, which might be the PHA production
pathway. Tafel plots also showed a marked shift in the redox
behavior of biocatalyst in concurrence to PHA production. With
the increase in time interval, bacteria exhibited a shift towards
reduction (0.2 V) during 12 h and 24 h. Behavior of biocatalyst to-
wards mid-point potential or near oxidation was observed during
Fig. 4. (a) Cyclic voltammograms derived with the function of different time
intervals using Bacillus tequilensis as biocatalyst; (b) and (c) Tafel plots derived from
cyclic voltammograms at different time intervals using GPES (4.0) software.
Table 1
Cyclic voltammetry, Tafel slope and polarization resistance of Bacillus tequilensis using spent wash efuents at OLR2 operation with the function of time intervals.
Time (h) Tafel slope (V/dec) Polarization resistance (R
p
, X)
Oxidation current (lA) Reduction current (lA) Oxidative (ba) Reductive (bc)
0 0.11 0.32 0.591 0.203 6.305
12 0.17 0.30 0.531 0.158 5.094
24 0.27 0.56 0.382 0.16 3.494
36 0.21 0.17 0.422 0.166 4.773
48 0.14 0.20 0.548 0.15 5.132
K. Amulya et al. / Bioresource Technology 158 (2014) 336342 341
36 h (0 V). By the end of operation, again a shift was observed to-
wards reduction (0.1 V). Higher reduction and near oxidation
behavior observed during 24 h and 36 h indicates higher PHA pro-
ductivity in accordance with the observed DH activity and sub-
strate degradation, which in turn strongly support the reported
lower polarization resistance and redox Tafel slopes.
4. Conclusion
The acidogenic SWE from anaerobic digestion pose serious
environmental problems if they are not appropriately handled be-
fore disposal. Therefore, the aim of this work was to investigate the
possibility of using SWE as a renewable resource for PHA produc-
tion using B. tequilensis. B. tequilensis showed the ability to store
PHA up to 40% of its DCW. Produced co-polymer contained higher
HB content than HV. Substrate degradation associated with reduc-
tion in acidication indicates the feasibility of this two stage pro-
cess for product recovery and concurrent waste valorization. This
integration strategy would not only utilize the VFA rich efuents
via a green, fruitful approach but would also contribute towards
reducing the PHA production cost.
Acknowledgements
The authors wish to thank the Director, CSIR-IICT for support
and encouragement in carrying out this work. Part of the research
was funded by CSIR in the form of XII ve year plan project on Sus-
tainable Waste Management Technologies for Chemical and Allied
Industries (SETCA; CSC 0113).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.
02.026.
References
Akaraonye, E., Keshavarz, T., Roy, I., 2010. Production of polyhydroxyalkanoates: the
future green materials of choice. J. Chem. Technol. Biotechnol. 85, 732743.
Albuquerque, M.G.E., Martino, V., Pollet, E., Avrous, L., Reis, M.A.M., 2011. Mixed
culture polyhydroxyalkanoate (PHA) production from volatile fatty acid (VFA)-
rich streams: effect of substrate composition and feeding regime on PHA
productivity, composition and properties. J. Biotechnol. 151, 6676.
Alkaya, E., Demirer, G.N., 2011. Anaerobic acidication of sugar-beet processing
wastes: effect of operational parameters. Biomass Bioenergy 35, 3239.
APHA, 1998. Standard Methods for the Examination of Water and Wastewater,
twentieth ed. American Public Health Association/American water work
Association/Water environment federation, Washington, DC, USA.
Bengtsson, S., Werker, A., Christensson, M., Welander, T., 2008. Production of
polyhydroxyalkanoates by activated sludge treating a paper mill wastewater.
Bioresour. Technol. 99, 509516.
Chakravarthy, K.B., Narasu, Lakshmi M., 2012. Production and optimization of lipase
from Bacillus tequilensis NRRL B-41771. Int. J. Biotechnol. Appl. 4, 134136.
Dinopoulou, G., Rudd, T., Lester, J.N., 1988. Anaerobic acidogenesis of a complex
wastewater: the inuence of operational parameters on reactor performance.
Biotechnol. Bioeng. 31, 958968.
Filipe, C.D.M., Daigger, G.T., Grady Jr., C.P.L., 2001. PH as a key factor in the
competition between glycogen-accumulating organisms and phosphorus
accumulating organisms. Water Environ. Res. 73, 223232.
Griebe, T., Schaule, G., Wuertz, S., 1997. Determination of microbial respiratory and
redox activity in activated sludge. J. Ind. Microbiol. Biotechnol. 19, 118122.
Hazer, B., Steinbuchel, A., 2007. Increased diversication of polyhydroxyalkanoates
by modication reactions for industrial and medical applications. Appl.
Microbiol. Biotechnol. 74, 112.
Jin, Y.X., Shi, L.H., Kawata, Y., 2013. Metabolomics-based component proling of
Halomonas sp. KM-1 during different growth phases in poly (3-
hydroxybutyrate) production. Bioresour. Technol. 140, 7377.
Khan, I., Gupta, P., Vakhlu, J., 2011. Thermo-alkaliphilichalotolerant detergent
compatible protease(s) of Bacillus tequilensis MTCC 9585. Afr. J. Microbiol. Res. 5,
39683975.
Kim, M.S., Kim, D.H., Cha, J., Lee, J.K., 2012. Effect of carbon and nitrogen sources on
photo-fermentative H
2
production associated with nitrogenase, uptake
hydrogenase activity, and PHB accumulation in Rhodobacter sphaeroides
KD131. Bioresour. Technol. 116, 179183.
Law, J.H., Slepecky, A.S., 1960. Assay of poly-b-hydroxybutyric acid. J. Bacteriol.
1960, 823836.
Lee, K.M., Gilmore, D.F., 2005. Formulation and process modeling of biopolymer
(polyhydroxyalkanoates: PHAs) production from industrial wastes by novel
crossed experimental design. Process Biochem. 40, 229246.
Lemos, P.C., Seram, L.S., Reis, M.A.M., 2006. Synthesis of polyhydroxyalkanoates
from different short-chain fatty acids by mixed cultures submitted to aerobic
dynamic feeding. J. Biotechnol. 122, 226238.
Luengo, J.M., Garcia, B., Sandoval, A., Naharro, G., Olivera, E.R., 2003. Bioplastics from
microorganisms. Curr. Opin. Microbiol. 6, 251260.
Ntaikou, I., Kourmentza, C., Koutrouli, E.C., Stamatelatou, K., Zampraka, A., Kornaros,
M., Lyberatos, G., 2009. Exploitation of olive oil mill wastewater for combined
hydrogen and biopolymer production. Bioresour. Technol. 100, 37243730.
Omar, S., Rayes, A., Eqaab, A., Viss, I., Steinbuechel, A., 2001. Optimization of cell
growth and poly(3-hydroxbutyrate) accumulation on date syrup by a Bacillus
megaterium strain. Biotechnol. Lett. 23, 11191123.
Passanha, P., Esteves, R.S., Kedia, G., Dinsdale, R.M., Guwy, A.J., 2013. Increasing
polyhydroxyalkanoate (PHA) yields from Cupriavidus necator by using ltered
digestate liquors. Bioresour. Technol. 147, 345352.
Santimano, M.C., Prabhu, N.N., Garg, S., 2009. PHA production using low-cost agro-
industrial wastes by Bacillus sp. Strain COL1/A6. Res. J. Microbiol. 4, 89
96.
Searmsirimongkol, P., Rangsunvigit, P., Leethochawalit, M., Chavadej, S., 2011.
Hydrogen production from alcohol distillery wastewater containing high
potassium and sulfate using an anaerobic sequencing batch reactor. Int. J.
Hydrogen Energy 36, 1281012821.
Sun, S., Guo, Z., Yang, R., Sheng, Z., Cao, P., 2012. Study on the triphenyltetrazolium
chloridedehydrogenase activity (TTCDHA) method in determination of
bioactivity for treating tomato paste wastewater. Afr. J. Biotechnol. 11, 7055
7062.
Vatsala, T., Raj, S., Manimaran, A., 2008. A pilot-scale study of biohydrogen
production from distillery efuent using dened bacterial co-culture. Int. J.
Hydrogen Energy 33, 54045415.
Venkata Mohan, S., Srikanth, S., 2011. Enhanced wastewater treatment efciency
through microbially catalyzed oxidation and reduction: synergistic effect of
biocathode microenvironment. Bioresour. Technol. 102, 1021010220.
Venkata Mohan, S., Mohanakrishna, G., Ramanaiah, S.V., Sarma, P.N., 2008.
Simultaneous biohydrogen production and wastewater treatment in biolm
congured anaerobic periodic discontinuous batch reactor using distillery
wastewater. Int. J. Hydrogen Energy 33, 550558.
Venkata Mohan, S., Venkateswar Reddy, M., Venkata Subhash, G., Sarma, P.N., 2010.
Fermentative efuents from hydrogen producing bioreactor as substrate for
poly(b-OH) butyrate production with simultaneous treatment: an integrated
approach. Bioresour. Technol. 23, 93829386.
Venkata Mohan, S., Venkateswar Reddy, M., Chandra, R., Venkata Subhash, G.,
Prathima Devi, M., Srikanth, S., 2013. Bacteria for bioenergy: a sustainable
approach towards renewability. In: Gaspard, S., Ncibi, M.C. (Eds.), Biomass for
Sustainable Applications: Pollution, Remediation and Energy. RSC Publishers
(ISBN 9781849736008).
Venkateswar Reddy, M., Srikanth, S., Venkata Mohan, S., Sarma, P.N., 2010.
Phosphatase and dehydrogenase activities in anodic chamber of single
chamber microbial fuel cell (MFC) at variable substrate loading conditions.
Bioelectrochemistry 77, 125132.
Venkateswar Reddy, M., Venkata Mohan, S., 2012a. Effect of substrate load and
nutrients concentration on the polyhydroxyalkanoates (PHA) production using
mixed consortia through wastewater treatment. Bioresour. Technol. 114, 573
582.
Venkateswar Reddy, M., Nikhil, G.N., Venkata Mohan, S., Swamy, Y.V., Sarma, P.N.,
2012b. Pseudomonas otitidis as a potential biocatalyst for polyhydroxy-
alkanoates (PHA) synthesis using synthetic wastewater and acidogenic
efuents. Bioresour. Technol. 123, 471479.
Venkateswar Reddy, M., Amulya, K., Rohit, M.V., Sarma, P.N., 2013. Valorization of
fatty acid waste for bioplastics production using Bacillus tequilensis: integration
with dark-fermentative hydrogen production process. Int. J. Hydrogen Energy.
<http://dx.doi.org/10.1016/j.ijhydene.2013.09.157>.
Wang, J., Yu, J., 2000. Kinetic analysis on inhibited growth and poly (3-
hydroxybutyrate) formation of Alcaligenes eutrophus on acetate under
nutrient-rich conditions. Process Biochem. 36, 201208.
Yu, H.Q., Fang, H.H.P., 2002. Acidogenesis of dairy wastewater at various pH levels.
Water Sci. Technol. 45, 201206.
Zhenhua, L.V., Yao, Y., Zhenmei, L.V., Min, H., 2008. Effect of tetrahydrofuran
on enzyme activities in activated sludge. Ecotoxicol. Environ. Saf. 70, 259
265.
342 K. Amulya et al. / Bioresource Technology 158 (2014) 336342