Bioprocess Biosyst Eng

DOI 10.1007/s00449-016-1603-z

ORIGINAL PAPER

Energetic assessment of fixation of CO2 and subsequent biofuel

production using B. cereus SM1 isolated from sewage treatment
plant
Somesh Mishra1 Suresh Gupta1 Smita Raghuvanshi1 Pratibha Pal1

Received: 22 December 2015 / Accepted: 3 April 2016

Springer-Verlag Berlin Heidelberg 2016

Abstract The ongoing work on global warming resulting

from green house gases (GHGs) has led to explore the
possibility of bacterial strains which can fix carbon dioxide
(CO2) and can generate value-added products. The present
work is an effort in this direction and has carried out an
exhaustive batch experiments for the fixation of CO2 using
B. Cereus SM1 isolated from sewage treatment plant
(STP). The work has incorporated 5-day batch run for
gaseous phase inlet CO2 concentration of 13 1 % (%v/
v). 84.6 (5.76) % of CO2 removal was obtained in the
gaseous phase at mentioned CO2 concentration (%v/v).
Energetic requirement for CO2 fixation was assessed by
varying Fe[II] ion concentration (0200 ppm) on the perday basis. The cell lysate obtained from CO2 fixation
studies (Fe[II] ion = 100 ppm) was analyzed using Fourier
transformation infrared spectroscopy (FTIR) and gas
chromatography-mass spectroscopy (GCMS). This analysis confirmed the presence of fatty acids and hydrocarbon
as valuable products. The hydrocarbons were found in the
range of C11C22 which is equivalent to light oil. The
obtained fatty acids were found in the range of C11C19.
The possibility of fatty acid conversion to biodiesel was
explored by carrying out the transesterification reaction.
The yield of biodiesel was obtained as 86.5 (0.048) %
under the transesterification reaction conditions. Results of
this research work can provide the valuable information in
the implementation of biomitigation of CO2 at real
scenario.

& Smita Raghuvanshi

smita@pilani.bits-pilani.ac.in
1

Department of Chemical Engineering, Birla Institute of

Technology and Science (BITS), Pilani 333031, Rajasthan,
India

Keywords CO2 fixation  Energetic assessment  Product

characterization  Transesterification  Material balance

Introduction
Increasing concentrations of greenhouse gases (GHGs)
caused by human activities is a major environmental concern. The concentration of carbon dioxide (CO2) was
reported as 401.16 ppm in the month of November 2015 by
Mauna Loa Observatory, Hawaii, USA [1]. The biological
route utilizing microalgae for CO2 mitigation and simultaneous value addition in the form of biofuels is identified
as one of the most viable options to tackle this problem [2].
However, the use of microalgae for CO2 sequestration is
limited due to various reasons: (1) unable to sustain at high
CO2 concentration, (2) lower oil productivity, (3) outdoor
cultivation, i.e., open pond system has lower CO2 fixation
efficiency, (4) high operating cost, (5) high nutrient
requirements, (6) availability of land to sequester CO2 at
large scale, and (7) difficulties in scale up of bioreactor
system [3, 4].
The limitations with respect to phototrophic CO2 utilization has motivated to look for more economic, efficient,
and effective biological alternative for CO2 mitigation and
utilization. The bacterial CO2 fixation got an immense
attention and advancements in the last decade. Bacterial
cells are smaller in size and may act as a prominent
replacement for algae by virtue of their fast growth rate and
ability to sustain high CO2 concentration [5]. Recent
studies have blurred the distinction between autotrophy and
heterotrophy, as heterotrophs are also capable of fixing
inorganic form of carbon via CO2 assimilation [6, 7]. One
study has shown that the microorganisms (heterotrophs)
such as Bacillus pumilus, Pseudomonas fragi, and

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Bioprocess Biosyst Eng

Micrococcus lylae are capable of sequestering CO2 and

converting them into carbonate [6]. Another study has
reported bacterial species, Serratia sp ISTD04 for CO2
mitigation and production of biofuels [7]. One more work
has been carried out for the CO2 sequestration for the
production of biosurfactant as value-added product using
Bacillius sp ISTS2 [8]. These reported studies were
focused on the characterization of bacterium strains which
have shown an ability to sequester CO2 and used sodium
bicarbonate (NaHCO3) as inorganic carbon source. However, actual cellular CO2 fixation efficiency and actual
assimilation of CO2 as biomass were not taken into account
in these studies. Though, these features are important in
order to estimate the energetic requirements for the batch
and continuous experiments and also for scale-up studies.
The evaluation of the process energetics not only enables to
understand the possible pathway for the reductive assimilation
of CO2 as cellular biomass but also helps in predicting the
possible yield of the desired product [9]. Nonphotosynthetic
microbes gain energy by catalyzing oxidation/reduction
reactions (redox reactions) that are thermodynamically
favored but kinetically inhibited. One of the energy sources
used by chemolithotrophic microorganisms is iron (Fe) which
is the second most abundant metal present in the lithosphere.
Naturally, iron is present in two oxidation states, Fe[II] and
Fe[III]. Its utilization as a cellular energy source depends on
the redox potential of the Fe[II]/Fe[III]. However, automatic
oxidation of Fe[II] to Fe[III] can also occur at pH values[5 in
oxic conditions [10]. The oxidation of Fe[II] is favored thermodynamically, and energy obtained from the oxidation of
Fe[II] to Fe[III] is used for the reduction of CO2 to organic
carbon [11]. Therefore, microbes have developed the alternative means of assimilating insoluble form of iron (Fe[III]) to
more soluble form of iron (Fe[II]) which include surface
reduction, lowering of pH, utilization of heme, or extraction of
protein-complexed metal and production of siderophores
(ferric ion-specific chelating agent) [12]. The utilization of
low soluble Fe[III] ion may require a specific mobilization
strategy, while an uptake of a soluble iron is facilitated by
high-affinity and low-affinity transport systems [13]. Therefore, microbes have developed different iron utilization
strategies to meet cellular requirement under varying environmental conditions. Thus, the energetic assessment of the
bacterial CO2 fixation not only gives overall insight and
possibility of the process but also encourages carrying out
more studies under real-time situations.
Biofuels derived from the microorganisms have a
promising future, as it has the ability to check the escalating fossil fuel cost and to replace them to an extent. Most
of the work for the production of biofuel is based upon
microalgae and cyanobacteria [14, 15]. There are limited
information about the use of bacteria for CO2 sequestration
to produce biodiesel (biofuel) and hydrocarbons. Biodiesel

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is defined as a fuel consisting of mono-alkyl esters of longchain fatty acids originating in fats, oils, or lipids.
Microbes are found to have a faster growth rate and are
capable to grow in large scales. This may be due to their
flexibility in metabolism, which gives them an advantage
over phototrophs for CO2 fixation and also the value
addition in terms of biofuel [5, 16].
The presence of complex enzyme fatty acid synthase
(FAS) in bacteria makes them capable of producing fatty
acids or fatty acids derivatives [17]. The fatty acids or fatty
acid derivatives are converted into nonmethane hydrocarbons by different pathways in bacterial cells [16, 18].
Experimental evidence suggested the accumulation of
lipids in bacterial cell ranging from 4 to 78.3 % of the total
cell dry weight [19]. Also, the microbial production of
long-chain hydrocarbons is having another possibility to be
used as a modern energy resource as they are the major
constituents of gasoline, diesel, and jet fuel [7].
The previous studies are mainly focused on the utilization of NaHCO3 as a source of CO2, and there are few
studies which have reported the energy assessment of CO2
fixation via chemolithotrophs. Hence, there is a scope of
study for the utilization of CO2 by bacterial species as a
carbon source and to have better understanding for energy
assessment of CO2 fixation.
In the present study, bacterial species are isolated from the
naturally selected mixed culture obtained from sewage treatment plant (BITS Pilani, Rajasthan, India). Biomitigation of
CO2 using isolated species by conducting the batch experiments is carried out. The study also focused on the thermodynamic assessment for CO2 utilization by isolated specie.
The feasibility of reductive cellular CO2 assimilation utilizing
Fe[II] as an electron source is estimated by calculating the
values of overall DGreaction of possible redox reaction at 25 C
and 1 bar [9]. The work is emphasized on the overall metabolic reactions rather than the stepwise reactions which constitute assimilatory processes including CO2 assimilation. The
work shows the oxidation of Fe[II] and the reduction of CO2
and is coupled with the assimilation of CO2 into biomass as
hexadecanoic acid. The obtained biomass and the leachate
were further analyzed for the identification of cellular products using FTIR and GCMS. Fatty acid methyl ester (FAME)
analysis of fatty acid products was also carried out to estimate
the yield of biodiesel formed.

Materials and methods

Sample collection site, chemicals, and culture
medium
Activated sludge was collected from the secondary clarifier
unit of the sewage treatment plant located at Birla Institute

Bioprocess Biosyst Eng

of Technology and Science (BITS) Pilani (28220 000 N,

75360 000 E), Rajasthan, India. The sample was transferred
to a sterile sample bottle and immediately brought to the
laboratory for further enrichment as described in the following section. The chemicals used in present study were
of analytical grade (HiMedia laboratories, Mumbai, India),
and the solutions were prepared using distilled water from
a Milli-Q system. Minimal salt medium (MSM) was prepared by dissolving following salts (g/L): K2HPO40.8,
KH2PO40.2, CaSO42H2O0.05, MgSO47H2O0.5,
and (NH4)2SO41.0 in distilled water and was autoclaved.
The pH of autoclaved culture media was found to be 6.7.
Thousand ppm stock solution of Fe[II] was prepared using
filter sterilization technique by dissolving 4.96 g of
FeSO4.7H2O in distilled water, and volume of the solution
was made up to 1 L. The pH of stock Fe[II] solution was
maintained as 3.5. The media for plating was prepared by
adding 2 % agar (%w/v) to nutrient broth (peptone5 g/L,
beef extract3 g/L, and NaCl5 g/L in distilled water).
Twenty milliliters of 10 N KOH and 24 N H2SO4 solutions
was prepared using distilled water from a Milli-Q system
for transesterification reactions.
Isolation and species identification
The sludge was mixed thoroughly with 100 mL of autoclaved distilled water in a beaker and kept for 4 h to allow
settling of sludge in sterile conditions. The 50 mL of settled sludge was mixed with equal amount of distilled water
and was kept for 5 min. The temperature of this study was
37 C. The obtained supernatant (2 mL) was mixed with
100 mL MSM in 500 mL volume of Erlenmeyer flask for
the further enrichment. The appropriate amount of stock
Fe[II] solution was added to maintain 50 ppm of Fe[II]
concentration in the 500-mL flask. The flask was sealed
from the top using the two-port assembly as shown in
Fig. 1. CO2air mixture (2 % (% v/v)) was then allowed to
pass through a capsule air filter at the flow rate of 1 L/min
(Fig. 1). The concentration of CO2 was monitored at the
outlet port at regular intervals of time. The ports were
closed, and the flask was airtight, when the concentration
of CO2 reached 2 % (%v/v) at the outlet. It ensured the
required concentration of CO2 in the gaseous phase. The
flask was kept in an orbital shaking incubator (MSW-132,
Macroscientific Work PVT., Ltd., India), and the culture
was grown at 37 C and 80 rpm for 3 days. After 3 days,
the 2 mL of grown culture was taken in place of supernatant and the same procedure was repeated for another
3 days by increasing the CO2 concentration from 2 % to
5 % (%v/v). The fully grown sixth-day culture at 5 % (%v/
v) CO2 concentration was used for the species isolation and
identification. Five different microbial species (SM1, 2, 3,
4, 5) were present in the culture obtained during CO2

fixation and were isolated using the streak plate method.

Batch experiments for 4 days were conducted for each
isolated species separately by maintaining 5 % (%v/v) inlet
CO2 concentration to check the CO2 fixation capability of
individual species. Gaseous and liquid-phase samples for
all experiments were analyzed on the per-day basis. Out of
the five isolated species, one of the best species was
selected on the basis of its CO2 fixing ability. The glycerol
stocks of selected isolate were prepared and preserved for
further studies.
Molecular identification and phylogenetic analysis
The plate of pure culture was prepared and sent to Xceleris
genomic labs Ahemdabad, India, for amplification of 16S
rRNA gene sequencing [20]. The obtained sequence was
analyzed by the BLAST-N tool available at the National
Center for Biotechnology Information (http://www.ncbi.
nlm.nih.gov). The taxonomic affiliation of the selected
isolate was assigned based on the closest match obtained
on pair-wise alignment in BLAST results. The 16S rRNA
gene sequence of related bacterial species was used for
construction of a phylogenetic tree using the neighborjoining (NJ) method by use of MEGA version 4.0 software
[21] and aligned using CLUSTAL-X. The pair-wise evolutionary distance was constructed by the neighbor-joining
method with bootstrap of 500 replicates to cluster the
associated taxa.
Batch study
The batch studies were carried out in a BOD incubator
shaker by maintaining shaking speed and temperature at
120 rpm and 37 C, respectively. Batch studies were performed in 20 flasks of 250 mL each and divided into four
groups. Each flask was added with 40 mL MSM and 2 mL
of enriched culture. The Fe[II] ion concentration was
maintained at 0, 50, 100 and 200 ppm in group 1, group 2,
group 3 and group 4, respectively. The flasks were sealed at
the top using a rubber stopper to avoid any loss of compounds present inside the flask. The batch experiments
were conducted at 13 1 % (%v/v) inlet CO2 concentration for the duration of 5 days. The initial CO2 concentration was selected on the basis of industrial CO2 emission
data [22]. The CO2 concentration was maintained in each
flask by following the similar procedure as described in
Isolation and species identification. One flask was taken
after every 24 h for the analysis of gaseous and liquid
samples. The control flask was kept without inoculums to
ensure that there was no loss of CO2 during the study and
also the bacterial isolate was responsible for the decrease in
CO2 concentration from the flask. All the experiments were
carried out twice to ensure the repeatability.

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Fig. 1 Schematic view of experimental setup

Biomass growth measurement and CO2 fixation rate

Biomass growth was reported in terms of the dry weight (g/L)
and optical density (OD). Plating was done on the per-day
basis to ensure the absence of contamination. Measurement of
dry weight was carried out on per-day basis by centrifugation
of the culture at the speed of 10,000 rpm for 20 min. Following centrifugation, the supernatant was discarded and the
obtained cells were kept at 80 C for 24 h to dry. The optical
density of bacterial culture was measured spectrophotometrically using an UV/Visible spectrophotometer (Evolution
201-Thermo Fisher Scientific, USA) at 600 nm. The relationship between optical density and the dry cell weight was
given by linear regression: y = 0.24124x ? 0.03928
(R2 = 0.97877). y in this equation is the biomass concentration (g/L), and x is the optical density (OD600). Therefore, the
optical density can be used to precisely predict the biomass
concentration. Maximum biomass productivity (PMax, g/L/d)
was calculated and given by Eq. (1):
PMax

Xt  Xo

tx  to

123

where Xt was the biomass concentration (g/L) at the end of

the cultivation period (tx) and Xo was the initial biomass
concentration (g/L) at the beginning of cultivation period
(to). Also, the maximum biomass concentration achieved
was designated as XMax.
Carbon dioxide fixation rate (RCO2 ) was calculated
according to the procedure described by S. Basu et al. [23].
The fraction of carbon (Cc) present in biomass estimated
according to the molecular formula (C6H12O7N) of the
biomass [24]. Cc was calculated as 0.34 and (RCO2 ) is given
by Eq. (2) as:
RCO2 Cc  PMax 

MCO2
MC

where MCO2 is the molecular weight of CO2 (g/mol) and

MC is the molecular weight of carbon (g/mol), respectively, and the CO2 removal rate (RRCO2 ) was given by
Eq. (3):
R:RCO2

g of CO2 fixed as C in biomass

 100
g of CO2 supplied initially

Bioprocess Biosyst Eng

Lipid and hydrocarbon extraction

The fifth-day culture of group 3 batch studies was used to
analyze the product formed during CO2 mitigation. The
culture was centrifuged at 15,000 rpm for 20 min, and the
cell pellet was obtained. The cell-free supernatant was
preserved for extracellular product characterization. The
cell lysis was carried via sonification and was utilized for
the intracellular product characterization. The extraction
was carried out by following the procedure described by
Swarnalatha et al. [25]. The nonpolar solvent system of
chloroform and methanol, mixed in a 2:1 ratio, was utilized
for the extraction. The cell lysate was diluted ten times
with the mixture of chloroform and methanol and kept on a
shaker at 160 rpm for 1 h to separate the phases. The
organic phase was concentrated on the rotary evaporator
and was further dissolved in chloroform for the analysis.
The cell-free supernatant was mixed with chloroform and
methanol (ratio 2:1) to extract the extracellular product.
Products obtained from cell lysis and cell-free supernatant
were kept over the shaker at 160 rpm for 1 h to ensure the
complete phase separation. After 1 h, the organic phases
(obtained from cell lysis and cell-free supernatant) were
concentrated on the rotary evaporator. The concentrate
obtained from both organic phases first mixture (chloroform and methanol) was redissolved in chloroform. Each
sample was preserved and transferred to 5 mL sample vials
for FTIR and GCMS analyses.
Analysis of extracted product (FTIR and GCMS)
The functional groups present in the samples obtained from
previous Lipid and hydrocarbon extraction were analyzed using FTIR (Perkin Elmer, Gladiator, USA). The
spectra were collected using PerkinElmer FTIR system
equipped with diffuse reflectance accessory with the range
of 4004000 cm-1. All spectra were acquired in transmission mode by the ATR and were plotted using the same
scale on the transmittance axis.
These samples were also analyzed to identify various
products formed during CO2 utilization by the isolated
species using gas chromatography-mass spectroscopy
(GCMS) (Model QP-2010 Plus, Shimadzu, Japan).
One microliter of each sample was analyzed by GC
MS. The capillary column DB-5 MS (0.25 lm film
thickness, 0.25 mm i.d., 30 m length) was used for the
gas chromatography. The initial oven temperature was
held at 50 C for 2 min, and injector and detector
temperature was maintained as 250 and 230 C,
respectively. The temperature was increased from 50 to
250 C at a rate of 10 C per min and was held for
3 min on reaching 250 C. The temperature was further
increased from 250 to 280 C at a rate of 15 C per min

and was held for 5 min on reaching 280 C. Helium

was used as a carrier gas (head pressure was 72.5 kPa,
and flow rate was maintained as 1.21 mL/min). The
conditions applied to MS analysis were as follows: scan
mode, start time of 4.00 min, end time of 31.99 min,
scan speed 1250, event time 0.5 s, start m/z 40.00, and
end m/z 650.00. Data were compared with the inbuilt
standard mass spectra library system (NIST-05 and
Wiley-8) of GCMS.
Transesterification and production of biodiesel
The products of cell lysate (after sonication) and
supernatant obtained from the batch study for fifth-day
sample (100 ppm Fe[II]) as per the method described in
the Lipid and hydrocarbon extraction were mixed
together for the transesterification reaction. The direct
method for transesterification reaction was carried out by
following the procedure of OFallon [26] in 25 mL
conical flask. The 2 mL of mixed sample was placed in
25 mL conical flask and mixed with 20 lL of methyl
tridecanoate which was used as an internal standard for
this study. Further, 1 mL of 10 N KOH along with 5 mL
of methanol was added in the same flask. The conical
flask was incubated in a water bath (55 C) for 1.5 h at
120 rpm. After incubation, the conical flask was cooled
below the room temperature in a cold tap water and it
was added with 1 mL of 24 N H2SO4. The conical flask
was incubated again at 55 C in water bath for 1.5 h at
120 rpm in order to complete the fatty acid methyl ester
synthesis (FAME) reaction. Once the FAME synthesis
was completed, the conical flask was cooled in a cold
tap water bath and mixture was transferred to the 50-mL
centrifuge tube. Twenty milliliters of hexane was added
to the existing FAME mixture in the centrifuge tube, and
it was vortex-mixed for 5 min on a multi-tube vortex.
The separation of phases was carried out by centrifugation for 5 min in a tabletop centrifuge. The layer of
hexane was concentrated on rotary evaporator, which
contained the FAME and was placed into a GC vial for
the further analysis. The final concentrated hexane phase
(rich in ester) was utilized to obtain product yield. The
yield was calculated in terms of the productivity of
FAME which was quantified using standard FAME mix
(C8C24) obtained from Supelco (USA) by following the
method described by Van Wychen and Laurens [27]. The
product yield was defined by the following Eq. (4)
Yield (%)

WFAME
 100
WBiomass

whereas WFAME is the weight of FAME obtained and

WBiomass is the weight of biomass taken.

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Bioprocess Biosyst Eng

Results and discussion

Species identification
Five isolated species were tested to check the CO2 mitigation capabilities at 5 % (%v/v) CO2 concentration. Out
of which isolate SM1 was found better as compared to
other species (SM1, 2, 3, 4, 5) and also has shown the
stable growth. Hence, isolate SM1 was selected for further
experimental studies. The molecular analysis based on 16S
rRNA gene homology has shown 99 % similarity with the
existing sequences of Bacillus cereus, respectively, in
NCBI database. The nucleotide sequences have been submitted to NCBI GenBank database with the accession no.
KJ755083 (Bacillus cereus SM1). Phylogenetic tree was
constructed using the gene sequence of the isolate, and the
representative bacterium of related taxa is shown in Fig. 2.
CO2 fixation by B. cereus SM1 at different Fe[II]
concentration
Flask studies have been conducted for isolated bacterium
for a period of 5 days in an elevated CO2 environment
{13 1 (% v/v)} at 0, 50, 100 and 200 ppm Fe[II] ion
concentration as an energy source. The control flask was
also kept without the bacterium inoculum by maintaining
the same CO2 and 100 ppm of Fe[II] concentration. Figure 3 shows the significant decrease in CO2 concentration
for 100 and 200 ppm of Fe[II] concentration for first 3 days
and thereafter became constant. The maximum CO2
removal efficiency for 100 and 200 ppm Fe[II] concentration was obtained as 84.6 (5.76) % and 80 (9.45) %,

respectively. This may be due to the availability of more

CO2 to be fixed by B. cereus SM1 and higher Fe[II] concentration (energy source) in first 3 days. The fate of Fe[II
concentration may be understood by the following explanation. Naturally, iron is present in two oxidation states,
Fe[II] and Fe[III]. The energy required for the CO2 fixation
by B.cereus SM1 [(chemolithotrophic species) is obtained
by a suitable oxidizing and reducing agent which in present
case is Fe[II] as an electron donor and O2 as an electron
acceptor] [28]. The biousable state is Fe[II] which gets
converted into Fe[III] during redox reaction. Fe[III] ion is
stable in oxic environment, but it has strong tendency to
hydrolyze to forms compounds that are often insoluble at
near neutral pH [10]. However, the presence of complexing
reagents can significantly increase the solubility of ferric
ion and is one of the strategy adopted by iron-utilizing
microbes [12]. Hence, at the end of the process, Fe[II] may
be present in form of Fe[II] (unutilized) and Fe[III] (produced by redox reaction). However, for 0 and 50 ppm of
Fe[II], no considerable decrease in CO2 concentration was
observed. The maximum CO2 removal efficiency at 0 and
50 ppm of Fe[II] was observed as 10.66 (1.23) % and
29.76 (0.44) %, respectively.
The control flask has not shown any change in CO2
concentration from the gaseous phase at the end of fifth day
(Fig. 3). The maximum CO2 removal efficiency from
gaseous phase was obtained as 84.6 [5.76] % at 100 ppm
of Fe[II] concentration. This study indicates that the optimum amount of energy source (100 ppm of Fe[II]) plays a
major role in CO2 fixation using B. cereus SM1.

25

Control
0 ppm Fe[II] ion concnetration (*)
50 ppm Fe[II] ion concnetration
100 ppm Fe[II] ion concnetration
200 ppm Fe[II] ion concnetration

CO2 concentration (% v/v)

20

15

10

0
1

Time (Days)

Fig. 2 Phylogenetic tree based on 16S rRNA gene sequences

showing the relationships between Bacillus between Bacillus cereus
SM1 and its related phylogenetic neighbors. The topology shown was
calculated with the neighbor-joining algorithm. Accession numbers
are indicated in brackets

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Fig. 3 CO2 fixation by B. cereus SM1 on per-day basis at varying

Fe[II] ion concentration. The data represent the mean SD. Statistically significant differences among duplicate groups, analyzed using
t test, are represented as p \ 0.05 denoted by (*)

Bioprocess Biosyst Eng

Biomass growth rate of B. cereus SM1

The biomass concentration in terms of dry weight (g/L)
vs. time (days) is shown in Fig. 4 for 0, 50, 100, and
200 ppm Fe[II] concentration. The dry weight for all
concentrations of Fe[II] was found to increase till day 5.
The maximum biomass concentration was found as 0.187
(0.003), 0.256 (0.008), and 0.386 (0.002) g/L at 50,
100, and 200 ppm of Fe[II] ion concentration, respectively (Fig. 4). The biomass observed at 50 ppm Fe[II]
concentration was 0.187 g/L which was nearly 73 and
48.33 % of the biomass obtained at 100 ppm and
200 ppm Fe[II] concentration, respectively. This indicates
that the increase in Fe[II] concentration from 50 to
100 ppm not only increases the CO2 removal efficiency
but also approximately doubles the biomass obtained.
Further increase in Fe[II] concentration from 100 to
200 ppm has shown an increasing trend in biomass productivity; however, no significant change in CO2 removal
efficiency from gas phase was observed. Thus, the above
result obtained describes that the increasing Fe[II] concentration up to a certain limit has enhanced the CO2
utilization; however, further increase has no significant
effect in CO2 utilization. This may be due to the fact that
the occurrence of Fe[II] or Fe[III] state is dependent on
the parameters such as pH, oxygen concentration, and
redox potential of the overall working system. Fe[II] ion
is found to be stable and is susceptible to spontaneous
chemical oxidation by molecular oxygen at low pH [10].
Fe[III] ion is stable in the presence of molecular oxygen
0.60

0 ppm Fe[II] ion concentration

50 ppm Fe[II] ion concentration
100 ppm Fe[II] ion concentration
200 ppm Fe[II] ion concentration

0.55
0.50

Dry weight (g/L)

0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00

Time (Days)

Fig. 4 Dry weight on per-day basis at different Fe[II] ion concentration. The data represent the mean SD. Statistically significant
differences among duplicate groups, analyzed using t-test, are
represented as p \ 0.05 denoted by (*) and at the 0.05 level
population variation are not significantly different

and has a tendency to hydrolyze easily. Due to this, the

bioavailability of Fe[II] is very low due to which bacterial
species have developed different Fe[III]-utilizing mechanisms [10]. Hence, there is a possibility of hydrolysis of
Fe[III] and more biomass formation at 200 ppm of Fe [II]
concentration. The R.RCO2 at 13 (1) % (% v/v) inlet
CO2 concentration for 0, 50, 100, and 200 ppm Fe[II] ion
concentration was obtained as 10.13 (0.006) %, 32.3
(0.0071) %, 44 (0.02) %, and 66.6 (0.0014) %,
respectively. This indicates that on increasing the Fe[II]
ion concentration from 100 ppm to 200 ppm also
increases the CO2 removal rate but has not much effect
on CO2 removal efficiency from the gaseous phase. It is a
well-known fact that dissolved inorganic carbon in culture
medium is comprised of CO2, HCO3-, and CO32- and the
microbes mainly utilizes HCO3- ion as an carbon source
[6]. Also, the solubility of CO2 in liquid media is given
by the equilibrium between volumes of CO2 dissolved to
its partial pressure in gaseous phase [28]. Therefore, in
the system it is very much possible that equilibrium is
achieved between the CO2(aq) and CO2(g) after dissolving respective amount of CO2 from gaseous phase. Thus,
the dissolved inorganic carbon in the form of HCO3- ion
was assimilated more as C in biomass at 200 ppm Fe[II]
ion concentration and hence increasing the R:RCO2 value
but does not have any significant effect on CO2 removal
efficiency from gaseous phase [29].
The microbial CO2 mitigation and extraction of valuable
biomolecule was known to be carried out majorly by
photosynthetic microorganisms such as algae and
cyanobacteria over the past decade. In one study, the
researchers have found that Chlorella sp. at 15 % (%v/v)
inlet CO2 concentration in sequential bioreactor has shown
the R:RCO2 value of 85.6 with PMax value of 0.95 in 8-day
run [30]. Another work which has used the mixed culture
containing species Chlorella sp., Scenedesmus sp., and
Ankistrodesmus sp at 5 % (% v/v) inlet CO2 concentration
in a vertical photobioreactor (batch operation mode) has
reported the RCO2 as 0.98 with PMax value of 4.90 and
R.RCO2 value of 59.80 % [31]. In another study, using algal
species Spirulina obliquus at 12 % inlet CO2 concentration
(% v/v) have reported the R:RCO2 value of 13.56 with PMax
value of 1.60, respectively, in serial tubular photobioreactor [32]. In present work, at 13 1 % inlet CO2 concentration (% v/v), the RCO2 value was found as
0.0638 0.003 and 0.0962 0.00071 with PMax value of
0.0512 0.002 and 0.0772 0.00056 in a 5-day batch
mode at 100 ppm and 200 ppm Fe[II] ion concentration,
respectively. The R:RCO2 values observed at 100 ppm and
200 ppm Fe[II] ion concentration are 44 (0.02) % and
66.6 (0.0014) %, respectively. Thus, the obtained results
indicate that the isolate strain B. cereus SM1 has an ability

123

Bioprocess Biosyst Eng

to grow well in high CO2 atmosphere ([10 % CO2 (%

v/v)) and hence can be seen as an promising candidate for
CO2 mitigation in absence of light. The biomass growth is
an important parameter which not only ensures the
microbial growth and also provides the important information in terms of value addition. Cell growth is associated
with two processes: (1) uptake of some material from the
cells environment and (2) release of metabolic end product
into the surroundings. These metabolic end products may
be considered for the value addition and also helps in
predicting the mechanism of CO2 fixation. The Liquid
phase product analysis (Lipids and hydrocarbons) discusses the products obtained from CO2 fixation.
Liquid-phase product analysis (Lipids
and hydrocarbons)
FTIR spectroscopy analysis
The products present in the cell lysate and supernatant
extract were analyzed for the determination of the structural features using FTIR. The purpose of FTIR study is to
get an understanding about which functional groups can be
present in the product. The information obtained from the
cell extract w.r.t chemical composition using FTIR helps in
the development of further characterization and purification strategy for value-added products obtained during
mitigation studies [33]. The liquid-phase FTIR spectrum of
the obtained products from cell lysate and supernatant
extracted with chloroform and methanol solvent system for
the present study are shown in Figs. 5 and 6, respectively.
The major peaks are shown at following wave numbers
3304.55, 2946.40, 2834.37, 1654.38, 1449.25, 1412.45,
1114.42, 1017.76, and 595.04 cm-1 for cell lysate extract.
The obtained peaks at 3271.27, 2900, 2800, 1645.34,

1410.65, 1241.92, 1014.05, 752.49, and 666.98 cm-1 represented the peaks obtained from supernatant extract. The
obtained spectrum band can be classified into two ranges
from 3600 to 2500 cm-1 and from 1700 to 1000 cm-1. The
presence of carboxylic acid group was indicated by
somewhat messy absorption pattern in the region of
33002500 cm-1 with the broad OH band superimposed
on the sharp CH stretching bands (2946.40, 2834.37, 2900
and 2800 cm-1) (Figs. 5, 6). The stretch 1654.38 and
1645.34 cm-1 indicated the presence of compounds having
C=O bonds (Figs. 5, 6). The exact position of this broad
band depends on whether the carboxylic acid is saturated or
unsaturated, dimerized, or has an internal hydrogen bonding [34]. The presence of peak at 1449.25 cm-1 in cell
lysate extract and supernatant indicated the presence of C
H bend of alkanes (Figs. 5, 6). The stretch at 1412.45 cm-1
for cell lysate extract (Fig. 5) and stretch at 1410.65 cm-1
for supernatant extract (Fig. 6) specified the presence of C
C stretch in rings, i.e., indicated the presence of aromatic
compounds. The peaks obtained at 1114.42 and
1017.76 cm-1 for cell lysate extract (Fig. 5) and
1014.05 cm-1 for supernatant extract (Fig. 6) have shown
the presence of compound having CO bond. The peak at
595.04 cm-1 for cell extract and 752.49 cm-1 and
666.98 cm-1 for supernatant extract indicated the CH
rock of long-chain alkanes [34]. Thus, FTIR spectrum of
both the cell lysate and supernatant extract indicated the
presence of compounds having C=O, CH, CO, OH
bonds. The analysis further indicated the presence of
compounds having aromatic ring and long-chain alkanes.
However, one has to take in consideration that presented
results can differ slightly when procedures of sample
preparation and especially cell growth conditions are
changed. The structural composition obtained from FTIR
results were further strengthened by carrying out Gas

90

2 8 3 4 .37
2946.4 0
60

1 65 4.3 8
1 44 9 .25 ,
1 41 2 .45

1 1 1 4 .42

3304.5 5
595.0 4

4 00 0

3 50 0

3 000

2 500

2 000

W aven u m b er (cm

15 00

-1

10 00

5 00

Fig. 5 FTIR spectrum of intracellular liquid phase of Bacillus cereus

SM1 in chloroform solvent

123

2800
80

2900
1645.34

1410.65
1241.92

60

3271.27

1014.05
752.49

40

1017.7 6

30

Transmittance (% T)

Transmittance (% T)

100

4000

3500

3000

2500

2000

1500

666.98

1000

500

-1

Wavenumber (cm )
Fig. 6 FTIR spectrum of extracellular liquid phase of Bacillus cereus
SM1 in chloroform solvent

Bioprocess Biosyst Eng

chromatography and mass spectroscopy (GCMS) studies

to get an understanding about which compounds can be
present following the above-mentioned functional groups.
GCMS analysis
Gas chromatography and mass spectroscopy (GCMS) is a
technique, which encompasses separation of various components through GC and detection of the separated components using MS. Table 1 shows the GCMS analysis of
various fatty acids present in cell lysate extract obtained
from fixation of CO2 using B. cereus SM1. The obtained
fatty acids were found in range of C11C19. The total fatty
acid present in cell lysate was obtained as 3.74 % of the
total cell extract. The bacterial cell is smaller in size
leading to the small percentage of fatty acid content as
compared to the eukaryotic cell. Table 2 shows the GC
MS analysis of various fatty acids present in supernatant
produced by the B. cereus SM1. The total fatty acid present
in supernatant was 16.24 % of the total extract. Fatty acids
are considered to be an integral component of cell membrane. They participate in various metabolic and regulatory
activities of the cell and also carry out the other functions
of cells. The metabolism of fatty acids has attractive
pathways and capable of producing metabolites similar to
high-energy petroleum distillates. Thus, the conversion of
fatty acids to fatty acid methyl ester (FAME) is an attractive pathway, which provides the value addition to the
process [35] and is also carried out in the present study as
shown in Transesterification and product yield.
The GCMS analysis of cell lysate extract also showed
the presence of hydrocarbons such as pentadecane, heptadecane, and nonane (Table 1). Pentadecane was found to
be 1.76 %, heptadecane as 0.66 %, and nonane as 0.13 %
of the total cell extract. The GCMS results of supernatant
extract showed the presence of hydrocarbons such as
decane (0.31 %), docosane (0.35 %), tetradecane (4.24 %),
pentadecane (1.62 %), and nonane (0.26 %) as reported in
Table 2. The total hydrocarbon production was comprised

of 6.28 % of the total extract. These hydrocarbons were

found to be present in extractable quantity. Bacterium is
well known for producing hydrocarbons [16]. The lower
molecular weight hydrocarbons are believed to permeate
through the cell membrane into the culture broth and can
be extracted and retained by the hydrocarbons present in
the bacterial cell which can further increase their carbon
number. The composition of kerosene oil, jet fuels, and
diesel is found in the range of C12C20 [16]. The species B.
cereus SM1 has also shown the production of hydrocarbon
in the range of C10C22 which is equivalent to light oil and
shows a potential as a valuable fuel [7] in the present study.
Thus, it can be said that the B. cereus SM1 can be categorized as one of those bacterial species which are capable
of producing the hydrocarbons.
Transesterification and product yield
The GCMS analysis mentions about the importance of
the FAME studies. In the present study, B. cereus SM1 was
tested for its potential to synthesize FAME by direct
transesterification process as described by OFallon [26].
The GCMS analysis of the cell lysate and supernatant
extract has indicated the presence of saturated as well as
unsaturated fatty acids in the sample as shown in Tables 1
and 2. The quality of biodiesel is determined by the length
of acyl chain, presence of unsaturation, and branching [36].
The biomass rich in monounsaturated fatty acids is desirable, but saturated fatty acids are also a necessary component for biodiesel [37]. The quantification of mixture for
both saturated and unsaturated FAME was difficult to
obtain. Hence, the fatty acids having carbon chain length in
the range of C14C18 (tetradecanoic acid to octadecanoic
acid) were taken as a target compound for obtaining the
product yield in the present study. The samples were
analyzed in duplicates, and average values of peak area
were used to generate the calibration plot. The calibration
plot for tetradecanoic methyl ester, hexadecanoic acid
methyl ester, octadecanoic acid methyl ester, and cis-9-

Table 1 GCMS analysis of cell lysate extract of Bacillus cereus SM1

S. no.

Run time

Formulae

Compound

% Area

Relative molecular
mass

Match
quality (%)

14.6

C11H14O3

Benzoic acid

0.76

194

89

19.017

C17H34O2

Hexadecanoic acid

0.29

270

89

20.717

C19H36O2

9-Octadecanoic acid (Z)-

0.97

296

86

20.9

C18H36O2

Heptadecanoic acid

0.22

284

86

12.979

C15H32

Pentadecane

1.76

212

95

14.2

C17H36

Heptadecande

0.66

240

92

15.4

C11H24

Nonane

0.13

156

87

123

Bioprocess Biosyst Eng

Table 2 GCMS analysis of extracellular products (supernatant) extracted in chloroformmethanol of Bacillus cereus SM1
S. no

Run time

Formula

Compound

% Area

Relative molecular
mass

Match
quality (%)

4.429

C5H10O2

Butanoic acid

4.03

102

95

14.698

C11H14O3

Benzoic acid

5.53

194

94

17.303

C14H28O2

Tetradecanoic acid

3.45

228

94

19.015

C18H36O2

Hexadecanoic acid

2.00

284

90

27.2

C21H42O2

Eicosanoic acid

0.41

326

73

27.483

C21H30O2

Dehydroabietic acid

0.35

314

67

7
8

7.359
7.942

C10H22
C22H46

Decane
Docosane

0.31
0.35

142
310

86
89

12.982

C14H30

Tetradecane

4.24

198

94

10

14.259

C15H32

Pentadecane

1.62

212

92

11

15.467

C11H24

Nonane

0.26

156

88

oleic methyl ester were plotted, and their respective coefficient of correlation (R2) was obtained. The relationships
for calibration plot are shown in Eqs. (5)(8).

Approximate material balance and thermodynamic

analysis

Y 12:83x  0:6; R2 0:922

Y 9:11x  0:56; R2 0:803

Y 6:3817x  0:466; R2 0:77

The gaseous phase utilization of CO2 by B. cereus SM1

was also proven by carrying out the approximate material
balance on the batch system. The material balance was
carried on the basis of elemental carbon (C) balance. The
total elemental carbon (C) balance is given by Eq. (9).

Y 17:55x  1:067; R2 0:81

FAME obtained in the present study was 0.09 mg

(0.002) tetradecanoic methyl ester, 0.25 mg (0.04)
hexadecanoic acid methyl ester, 0.12 mg (0.02) octadecanoic acid methyl ester, and 0.098 mg (0.01) cis-9-oleic
methyl ester. The total FAME found in the present study
was obtained as 86.5 mg (0.048) from 100 mg of biomass. Thus, the product yield of 86.5 (0.048) % from
fixation of CO2 by B. Cereus SM1 was obtained. In the
previous study reported in the literature, thirty bacterial
strains for producing fatty acids for biofuel production was
investigated which revealed that five of the isolates produced biofuel with product yields higher than 80 % and the
maximum yield of 93.2 % [37]. Another work has also
carried out the FAME analysis for biofuel production by
using base-catalyzed transesterification reaction [7]. The
maximum yield was obtained as 96 % at 40 C, with 1 %
NaOH (as a catalyst) and 3-h reaction time. In present
work, the product yield was obtained as 86.5 (0.048) %,
which is comparable with the above-reported studies.
Therefore, the isolate bacterium B. cereus SM1 can be
categorized as oleaginous microbes which has shown the
potential for the production of bio-oil. Thus, the present
work has energetically assessed the biomitigation of CO2
from the gaseous phase by B. cereus SM1 and simultaneous shows the potential for production of biodiesel and
hydrocarbons that can be utilized as biofuel.

123

MCin MC;go MC;bo Dissolved C as (aq) CO2

where MCin is mass of C supplied as CO2, MC,go is C left in

gaseous phase as CO2 after fifth-day batch, and MC,bo is C
assimilated as biomass.
The mass of C in the form of CO2 supplied to the system
was calculated by ideal gas law and was obtained as
0.0123 g. By considering 84.6 % fixation, the amount of C
present in gaseous phase was obtained as 0.00189 g. Mass
of C in biomass was estimated based on the molecular
formula of biomass (C6H12O7N) [24]. The biomass
obtained for 84.6 % removal at 100 ppm Fe[II] concentration was 0.0132 g. Based on the molecular formula of
biomass, the mass of C in biomass was estimated as
7.26 9 10-3 g. The dissolved C in aqueous phase was
calculated using Henrys law at the end of fifth day. The
dissolved C was obtained as 2.65 9 10-3 g at 0.13 atm
partial pressure. The values were substituted in Eq. (9).
L.H.S. and R.H.S. of Eq. (9) were calculated and were
obtained as 0.0123 g and 0.0118 g, respectively. These
values of Cin and Cout were approximately same which
indicated that the gaseous phase CO2 is mainly fixed by the
biological activity of B. cereus SM1.
Energy required to fix available CO2 by in the batch
system was also estimated and compared with the available
energy in terms of Fe[II] ion. The thermodynamic feasibility study was carried out at 100 pm Fe[II] as it was

Bioprocess Biosyst Eng

showing the maximum removal efficiency of CO2 [84.6

(5.76)] % as shown in CO2 fixation by B. cereus SM1 at
different Fe[II] concentration. The B. cereus SM1
obtained metabolic energy from the aerobic oxidation of
Fe[II] to Fe[III] and the reduction of Fe[III] to Fe[II] with
H2 as represented by Rxns (1) and (2).
3Fe2 0:5 O2 3 H2 O ! 3Fe3 6 H

3Fe3 H2 6 H ! 3Fe2 4H2 O

Overall reaction of reduction of CO2 into cellular biomass as hexadecanoic acid is given by Rxn (3).
16 CO2 Fe2 90 e 91 H
! C16 H30 OOH Fe3 30 H2 O

Raw material

Unit

Cost (Rs)

CO2 cost

Rs/kg

Water cost

Rs/kg

Nutrient cost

Rs/g

4.790

Production days

1g

396.4

Total raw material cost

Product cost (hexadecanoic acid ([99 %))
EP

4.790
22.0786

Equation to estimate Gibbs free energy of reaction is

given by Eq. (10).
DGreaction DGproduct  DGreactant

Table 3 Cost of raw materials utilized and product obtained during

the batch process

10

Gibbs free energy change in product and reactant were

calculated using the values standard Gibbs free energy of
formation of various compounds as reported by Thauer
et al. [24]. The Gibbs free energy of product and reactant
was obtained as -7425.6 and -6255.19 kJ mol-1,
respectively. This results in the value of the Gibbs free
energy of reaction as -1170.41 kJ mol-1. Therefore, the
energy required to fix 6.05 9 10-4 mol of CO2 as hexadecanoic acid was estimated was 0.0443 kJ which is less
as compared to the energy available (0.133 kJ for 100 ppm
of Fe[II]) in form of Fe[II]. Hence, thermodynamic analysis
also confirms the fixation of gaseous CO2 by B. cereus
SM1 using Fe[II] as energy source.
Feasibility of the process
The feasibility of the process is demonstrated by calculating the economic potential (EP) of the process as described
in the literature [38]. The economic potential is calculated
by Eq. (11) as:
EP Product cost  Raw material cost  Operating cost
11
In the present study, hexadecanoic acid was considered
as the main product obtained for deducing the economic
potential of the process. Operating cost considered to have
no effect on the cost of batch process [38] and hence taken
as 0 in the present study. The raw material and their
associated costs are given in Table 3. The weight of
nutrient utilized was 2.55 g, and hence, the cost associated
is Rs 12.21. The weight of hexadecanoic acid obtained was
86.5 mg, and associated cost is Rs 34.28. The cost of
chemicals is taken from HiMedia catalogue (2015), and
hexadecanoic acid cost is taken from catalogue of Sigma-

Aldrich. Therefore, according to Eq. (11), the EP was calculated as Rs 22.0786. Thus, the positive value obtained for
Ep shows that the process is very much feasible and can be
scaled up as the economic and effective solution for CO2
biomitigation.

Conclusions
The present work demonstrates the efficacy of mitigation
of CO2 using B. cereus SM1 in a batch mode. The
maximum removal efficiency was obtained as 84. 6
(5.76) % in the gaseous phase for 13 1 % (% v/v)
inlet CO2 concentration at 100 ppm of Fe[II]. This study
concluded that the optimum amount of energy source
(100 ppm of Fe[II]) plays a major role in CO2 fixation
using B. cereus SM1. The presence of value-added
products such as fatty acids and hydrocarbons is confirmed in the extracts obtained from cell lysate and
supernatant. The total FAME was obtained as 86.5 mg
from 100 mg of biomass showing the product yield of
86.5 (0.048) %. The FAME analysis supported that the
isolated strain B. cereus SM1 falls into the category of
oleaginous microbes. The production of biodiesel through
transesterification reaction has shown the importance of
B. cereus SM1 for the industrial purpose. The utilization
of gaseous phase CO2 by B. cereus SM1 is proven by the
approximate material balance and energy balance for the
present CO2 fixation system. The material balance and
energetic assessment shown for the present work could
enable to facilitate the implementation of such studies
under real-time scenarios.
Acknowledgments Authors are thankful to Council of scientific and
industrial research (CSIR) for providing the scholarship to the PhD
student (09/719 (0061)/2013 EMR-I), Department of Science and
Technology (DST), New Delhi and University Grants Commission
(UGC), New Delhi, India, for providing facilities to carry out the
research work.

123

Bioprocess Biosyst Eng

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