Fuel 159 (2015) 189196

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Fuel
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Development of semi-synthetic microbial consortia of Streptomyces

coelicolor for increased production of biodiesel (fatty acid methyl esters)
Shashi Kant Bhatia a, Da-Hye Yi a, Yong-Hyun Kim a, Hyun-Joong Kim a, Hyung-Min Seo a, Ju-Hee Lee a,
Jung-Ho Kim a, Jong-Min Jeon a, Kyoung-Soon Jang b, Yun-Gon Kim c, Yung-Hun Yang a,d,
a

Department of Microbial Engineering, College of Engineering, Konkuk University, Seoul, Republic of Korea
Biomedical Omics Group, Division of Bioconvergence Analysis, Korea Basic Science Institute, Cheongju 363-883, Republic of Korea
Chemical Engineering, Soongsil University, 511 Sangdo-dong, Seoul 156-743, Republic of Korea
d
Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University, Seoul 143-701, Republic of Korea
b
c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 S. coelicolor accumulate fatty acids up

to 3.2% of biomass.
 S. coelicolorDmanA: R. eutropha

produce 124% more fatty acids as

compared to S. coelicolor.
 The CN value of biodiesel produced by
consortia is 64.56.
 Oxidation stability of biodiesel is
75.98 h.

a r t i c l e

i n f o

Article history:
Received 14 February 2015
Received in revised form 23 June 2015
Accepted 25 June 2015
Available online 30 June 2015
Keywords:
Biodiesel
Consortium
Streptomyces coelicolor
Ralstonia eutropha
Triacylglycerol

a b s t r a c t
Biodiesel, an alternative to petroleum oil has gained signicant attention from the research community
because of its high energy content and good compatibility with existing engine systems. It can be produced from many different sources, such as animals, plants, and microbes. In this study, we demonstrated
the overproduction of fatty acid methyl esters (FAMEs) using a synthetic consortium of manA mutant
Streptomyces coelicolor with Ralstonia eutropha. The synthetic consortium of S. coelicolorDmanA: R. eutropha produced 114 mg/L fatty acids, which is 124% higher than the amount produced using S. coelicolor
alone. Overall, the fatty acids produced by the consortia S. coelicolorDmanA: R. eutropha were composed
of medium chain fatty acid (MCFA): long chain fatty acid (LCFA): very long chain fatty acid (VLCFA) in
8.75: 91.0: 0.25 proportion, and contained 75% saturated and 25% unsaturated fatty acids, which resulted
in FAMEs with better cetane number (65) and oxidation stability (76 h) than the fatty acids produced by
one strain alone. Nile red staining and subsequent uorescence spectroscopy revealed S. coelicolorDmanA
as good candidate for triacylglycerol (TAG) accumulation. Phospholipid-derived fatty acids (PLFA) analysis of consortia shows that S. coelicolorDmanA and R. eutropha synergistically support each others growth.
The results suggest that the synthetic consortium provides an approach for biodiesel production along
with improved quality.
2015 Elsevier Ltd. All rights reserved.

Corresponding author at: Department of Microbial Engineering, College of

Engineering, Konkuk University, Seoul, Republic of Korea. Fax: +82 2 3437 8360.
E-mail address: seokor@konkuk.ac.kr (Y.-H. Yang).
http://dx.doi.org/10.1016/j.fuel.2015.06.084
0016-2361/ 2015 Elsevier Ltd. All rights reserved.

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S.K. Bhatia et al. / Fuel 159 (2015) 189196

1. Introduction
Fossil fuels, such as mineral oil and coal have played major roles
as energy sources in human development and industrialization,
and their demand is increasing day by day. There is a need to
increase fossil fuel production to fulll the growing humanitarian
obligations, but experts have opined that the production of those
energy resources has already peaked, and thus it is expected that
the increasing demands cannot be met [1]. To solve those urgent
issues, the use of renewable energy sources, especially biodiesel,
needs to gain more widespread acceptance [2].
Biodiesel is made up of mono-alkyl esters of long chain fatty
acids, and is a promising alternative fuel that can be used without
making changes to preexisting diesel engines [3]. Today, fatty acids
are the most-common constituents of biodiesel, and can be
extracted from plants and microbes [4]. However, biodiesel production has many limitations, including its availability, competitive pricing, and posing a threat to reserve food stock [5,6].
The use of oleaginous microbes for biodiesel production provides
an attractive solution to overcome these limitations, as it is renewable, has a short production cycle, and is easier to scale up [7].
Prior to conversion to biodiesel via esterication, microbial fatty
acids need to be separated from cells through a series of
energy-intensive steps, such as cell harvesting, drying, and solvent
extraction [3]. In bacteria, only a few examples of substantial TAG
accumulation have been reported, such as Mycobacterium,
Nocardia, Rhodococcus, Streptomyces, Bacillus and Shewanella [814].
The properties of the biodiesel are determined by the amount
and type (chain length and double bonds) of each fatty acid present
in the mixture [15,16]. Microbial consortia engineering (MCE) has
become a well-established tool in biotechnology. The interactions
between members of a consortia typically enables the efcient
use of resources through the foundation of many cooperative interactions, and the division of labor through functional differentiation
and specialization [17,18].
Different types of consortia have been applied, including articial microbial consortia (AMC) (composed of two or more
wild-type populations whose interactions do not typically occur
naturally), synthetic microbial consortia (SMC) (system of
metabolically engineered microbes which are modied through
genetic engineering to establish an interaction), and natural microbial consortia (NMC) which exist in natural environments [19].
Streptomyces coelicolor can produce two pigmented antibiotics,
red tripyrolle undecylprodigiosin (RED) and blue polyketide actinorhodin (ACT) [20]. The S. coelicolorDmanA mutant, previously
generated in this laboratory by an insertional inactivation of
manA (SCO3025), which encodes phosphomannose isomerase
(PMI). The mutant exhibited a bld-like phenotype, i.e., fail to form
aerial hyphae and antibiotic production (blue actinorhodin and red
tripyrolle undecylprodigiosin) in liquid media [21]. This reduction
in antibiotics production by S. coelicolorDmanA mutant can be
channeled into TAG production as antibiotic and TAG synthesis
pathways are interrelated [20].
Inspired by the widespread occurrence of synergistic microbial
communities in nature, an innovative alternative approach was
explored herein: the design and construction of a microbial consortium consisting of different species that cooperate to achieve the
direct conversion of biomass (starch) to biodiesel. For the construction of a consortium and subsequent fatty acid production, screening of microbial specialists, including S. coelicolor, Shewanella
oneidensis, Bacillus pumilus, and Ralstonia eutropha was performed.
S. coelicolor and Bacillus sp. hydrolyzes starch and can use free
sugar as carbon source [17,18], while R. eutropha and S. oneidensis
both are unable to utilize starch and component monosaccharides
(glucose) as carbon source [22,23]. Various combination of these
microbes were tried to ensure carbon supply and accumulation

of fatty acids. The microbial consortia made up of S.

coelicolorDmanA: R. eutropha was established, where S. coelicolor
hydrolyzed the starch, accumulated the fatty acids, and released
organic acids, which were further used by R. eutropha as carbon
source. R. eutropha helps to modulate the media pH favorable for
S. coelicolor growth, and allowed the production of biodiesel with
an improved cetane number and oxidation stability.
2. Material and methods
2.1. Chemicals
The fatty acids methyl esters (FAMEs) standards (RM-5 and
Rapeseed mix) and chloroform/methanol (derivatizing reagent)
were purchased from SigmaAldrich (St Louis, MO, USA). The sulfuric acid (conc.) was purchased from Merck (Darmstadt,
Germany). Other chemicals for media were procured from BioM
Laboratories (Cerritos, USA).
2.2. Microorganisms and cultivation conditions
In order to study biodiesel production, the following microorganisms were used: S. coelicolor A3(2) M145 that was purchased
from the Korean Culture Type Collection (KCTC, South Korea);
and B. pumilus (ATCC 7061), S. oneidensis MR-1 (ATCC 700550),
and R. eutropha H16 (ATCC 17699) which were procured from
the American Type Culture Collection (ATCC). The mutant,
Streptomyces coelicolorDmanA used in this study, was previously
produced in this laboratory to study the phosphomannose isomerase (manA) deletion effect on antibiotic production in S. coelicolor [21]. Minimal salt media M9, with 0.1% yeast extract and
1% glucose, was initially used for the screening of bacteria for triacylglycerol (TAG) accumulation, and after that for consortia
growth, instead of glucose, 1% starch was used as a carbon source.
S. coelicolor spores were cultivated on R5 agar plates for 72 h, harvested by scraping, suspended in 20% (v/v) glycerol and stored at
80 C for further use. S. coelicolor seed culture was prepared by
inoculating spores in a 50 mL LB liquid medium with ve glass
beads of 3 mm diameter, and incubated at 30 C under shaking
condition (200 rpm). The germinated spores were harvested by
centrifugation (3200g, 4 C, 10 min) and resuspended in 5 mL
ion-free water. The 0.1 mL (2  106 CFU) germinated seed culture
was used as inocula. All microbes were precultured on a 5 mL scale
in a LB media and used as seed cultures. The microbes were cultured individually (0.1 mL seed culture) and for the consortia in
combination of two, three, and four microbes using 1:1, 1:1:1,
and 1:1:1:1 ratios seed culture, respectively, thereby keeping the
nal inoculum volume constant (0.1 mL). The microbes were cultured on a 10 mL scale in tubes with a capacity of 25 mL at 30 C,
with the agitation at 160 rpm for 72 h, after which an analysis
was done for the TAG accumulation.
2.3. Analytical method
The GC/MS chromatography was used for the detection and
quantication of fatty acids, according to the method described
previously, with a slight modication [24,25]. For methanolysis
of the fatty acids, approximately 10 mg of freeze-dried cells were
weighed and placed in Teon-stoppered glass vials, then 1 mL
chloroform and 1 mL methanol/H2SO4 (85:15 v/v) were added to
the vials, after which they were incubated at 105 C for 2 h, cooled
to room temperature, and then incubated on ice for 10 min. After
adding 0.5 mL of ice cold water the samples were thoroughly
mixed by vortexing for 1 min, and then centrifuged at 2000g. The
organic phases (bottom) were extracted by pipette and moved to

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S.K. Bhatia et al. / Fuel 159 (2015) 189196

clean borosilicate glass tubes, containing Na2SO4. The GCMS chromatography was then performed with a Perkin Elmer Clarus 500
Gas Chromatograph that was connected to a Clarus 5Q8S Mass
Spectrometer at 70 eV (m/z 50550; source at 230 C and quadruple at 150 C) in the EI mode with an Elite 5 ms capillary column
(30 m  0.25 mm i.d.  0.25 lm lm thickness; J&W Scientic,
USA). The carrier gas, helium, was used at the ow rate of
1.0 mL/min. The inlet temperature was maintained at 300 C, and
the oven was programmed to start at 150 C for 2 min before
increasing to 300 C at 4 C/min, in which the temperature was
maintained for 20 min. The injection volume was 1 lL, with a split
ratio of 50:1. The structural assignments were based on an interpretation of the mass spectrometric fragmentation and conrmed
by comparison with the retention times and fragmentation patterns of the authentic compounds and spectral data that was
obtained from the online libraries of Wiley (http://www.palisade.
com) and NIST (http://www.nist.gov). The internal standard used
was ten microlitre of methyl heneicosanoate (10 mg/mL). The
volatile organic acids present in the culture media were monitored
by high-performance liquid chromatography (Agilent, CA, USA),
using an Aminex HPX-87H column (Bio-RAD, CA, USA) at 50 C,
with a diode array detector at a wavelength of 210 nm, using
5 mM sulfuric acid as the mobile phase, isocratically, with a ow
rate of 0.6 mL/min, as previously described [26].
2.4. Screening of microbes for TAG synthesis
The different microbes were individually screened for fatty
acids accumulation, under the culture conditions that were given
above. After 72 h, the cultures were centrifuged at 4000g to separate the cells, and further analyzed for the TAG after methanolysis.
S. coelicolor, a known accumulator of fatty acids was further used to
construct synthetic consortia with the screened microbes, and
various microbial consortia were constructed, i.e., S. coelicolor:
B. pumilus (SC:BP), S. coelicolor: R. eutropha (SC:RE), S. coelicolor:
S. oneidensis (SC:SO), S. coelicolor: B. pumilus: S. oneidensis
(SC:BP:SO), S. coelicolor: B. pumilus: R. eutropha (SC:BP:RE),
S. coelicolor: R. eutropha: S. oneidensis (SC:RE:SO) and S. coelicolor:
R. eutropha: S. oneidensis: B. pumilus (SC:RE:SO:BP). The SC:RE in
combination, demonstrated the highest amount of TAG production,
compared with the other constructed synthetic consortia. To
further improve the fatty acids accumulation, a mutant
S. coelicolorDmanA (SCDmanA) was used in combination with RE.
2.5. Calculation of biodiesel properties
The focus of this work was to screen and develop a synthetic
microbial consortia for use in biodiesel production. The important
chemical and physical properties of the biodiesel were calculated
from the FAMEs composition, including the following: saturated
fatty acid (SFA), monounsaturated fatty acid (MUFA), polyunsaturated fatty acid (PUFA), degree of unsaturation (DU), saponication
value (SV), iodine value (IV), cetane number (CN), long chain saturated fatty (LCDF), cold lter plugging point (CFPP), cloud point
(CP), allylic position equivalent (APE), bis-allylic position equivalent (BAPE), oxidation stability (OS), higher heating value (HHV),
kinematic viscosity (l), and density (q). The fuel properties can
be calculated directly from the FAMEs proles [13,27]. Herein,
the Biodiselanalyzer v1. 1 software (publicly available at http://
www.brteam.ir/biodieselanalyzer) was used for the calculation of
the aforementioned properties [28].
2.6. Nile red staining for lipid accumulation
The Nile red staining method [29] was used, with a slight modication of the previously described method for lipid accumulation

study. A stock solution of Nile red (1 mg/mL) was prepared in acetone and stored in the dark to protect it from light. The staining, of
the SCDmanA, RE and SCDmanA:RE consortia, was performed by
incubating 200 lL culture with Nile red (50 lg/mL) and 5% DMSO
(v/v) to improve the permeability of the cells for 18 h at room temperature. In order to analyze the lipid accumulation, uorescence
spectroscopy was performed. For spectroscopy, the samples were
analyzed immediately after staining using a Perkin Elmer LS-55
uorescence spectrometer.
2.7. Consortia population dynamics
Different microbial groups have different phospholipid-derived
fatty acids (PLFA), which can be used as a marker to study microbial communities. The actinomycetes are represented by methylated fatty acids (10Me16:0, 10Me17:0, and 10Me18:0), while
the Gram-negative bacteria are represented by the cyclopropane
fatty acids (cy17:0, 16:1x7, 18:1x7, and 17:1x9) [30]. PLFA analysis can be used to study the population dynamics of the consortia
SCDmanA:RE, as these are actinomycetes and Gram-negative bacteria, respectively. PLFA extraction was performed using a 10 mL
consortia culture, centrifuged at 4000g for 10 min at 4 C to remove
the media components, the cell pellet was further washed with
ion-free water two times. The cell pellet was freeze-dried overnight and further subjected for PLFA extraction by adding 1 mL
chloroform. The chloroform phase was collected, reduced in volume by rotary evaporation, and fractioned by chromatography on
silicic acid (mesh size: 200400). The neutral lipids and glycolipids
were eluted with chloroform (5 mL) and acetone (5 mL), respectively, and discarded. The polar lipids were eluted with (5 mL)
methanol and collected in glass test tubes. The solvent was evaporated under a stream of oxygen-free nitrogen, and the residue subjected to alkaline methanolysis by vortexing for 30 s in a mixture
(2 mL) of 0.56% (w/v) KOH in dried methanol (BDH) and a toluene/methanol solution [1:1 (v/v)], followed by incubation at 37 C
for 30 min. After cooling, the mixtures were neutralized with
0.3 mL of 1 M acetic acid and fatty acid methyl esters (FAMEs) were
extracted using 2 mL of hexane and water. The upper layer was collected and after evaporation of the solvent under oxygen-free
nitrogen, the FAMEs were resuspended in chloroform. The FAMEs
were quantied by GCMS, as mentioned in the above
Section 2.2. The ratio of the SCDmanA:RE was calculated from
the PLFA marker, 10M16:0 and cy17:0 concentration, which were
present in the SCDmanA and RE respectively, and the standard
used was a pure culture PLFA amount (SC and RE). The microbial
interaction study was also performed using an organic acid prole,
pH and lipid accumulation study.
3. Results and discussion
3.1. Screening of microbes
Various microbes were screened for fatty acids accumulation, as
summarized in Table 1. The fatty acid accumulation in

Table 1
Fatty acid composition and productivity proles of different microorganisms.
Parameter

SC (%)

RE (%)

SO (%)

BP (%)

MCFA
LCFA
VLCFA
SFA
UFA

5.1
93.7
1.04
75.63
24.36

5.3
92.3
2.29
37.21
62.79

1.60
96.96
1.43
92.34
7.65

10.72
87.62
1.68
85.61
14.41

Total content (%)

3.2

1.2

3.0

0.4

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S.K. Bhatia et al. / Fuel 159 (2015) 189196

Streptomyces sp., Bacillus sp. and Shewanella sp. was conrmed in

this study, as it has already been reported [1114]. Among those
tested microbes, SC demonstrated comparable biomass production, i.e., 1.6 g DCW/L, along with 51.0 mg/L (3.2%) fatty acids production. The fatty acids in SC were composed of 76% saturated fatty
acid and 24% unsaturated fatty acid. BP also showed good biomass
production (1.3 g/L), but very little fatty acid accumulation, i.e.,
0.4% (5.0 mg/L). The other two microbes which were examined,
RE and SO showed little growth with an accumulation of 1.4%
(5.7 mg/L) and 3.0% (21.2 mg/L) fatty acids, respectively (Fig. 1).
RE and SO are poor in glucose utilization [22,23], exhibited little
biomass production and fatty acids accumulation. RE, total fatty
acids were composed of mostly unsaturated fatty acids (63.0%),
and with only 37.0% saturated fatty acids. Although, RE, SO, and
BP were not found to be good accumulators of fatty acids, but in
order to enhance the fatty acid production, they were tested for
the construction of various consortia with SC.
3.2. Synthetic consortia for increased fatty acid accumulation
SC has amylase activity and can utilize starch as a carbon source
[31]. All the microbes investigated for fatty acid accumulation
were used to prepare synthetic consortia with SC, in order to
increase the biomass production and improve fatty acid accumulation. Seven different consortia using four microbes were tested
(Fig. 2). Increase in the biomass production and fatty acid accumulation were observed for all the combinations of consortia. Among
them, the synthetic combination of SC:RE was found to be the best
combination for increased fatty acid accumulation (86.0 mg/L),
with 2.0 g/L biomass production. The other synthetic consortia of
SC, i.e., SC:BP, SC:BP:RE, and SC:RE:SO, also showed enhancement
in the biomass production, with 55.0%, 59.1% and 66.5% more fatty
acid accumulation, respectively. The other consortia were not
found to be a better combination to allow increased accumulation
of fatty acids, although improvement in biomass production was
observed.
3.3. Strategy for the enhancement of FAMEs production

Fig. 2. Biomass and fatty acid methyl ester proles of different consortia. Different
combinations of microbes were used for consortia construction, as follows:
S. coelicolor: B. pumilus (SC:BP), S. coelicolor: R. eutropha (SC:RE), S. coelicolor:
S. oneidensis (SC:SO), S. coelicolor: B. pumilus: S. oneidensis (SC:BP:SO), S. coelicolor:
B. pumilus: R. eutropha (SC:BP:RE), S. coelicolor: R. eutropha: S. oneidensis (SC:RE:SO),
and S. coelicolor: R. eutropha: S. oneidensis: B. pumilus (SC:RE:SO:BP).

fatty acid content of SCDmanA, compared with SC was due to

defects in antibiotic production [20]. SC which can produce two
pigmented antibiotics, red tripyrolle undecylprodigiosin (RED)
and blue polyketide actinorhodin (ACT), is known to have multiple
pathways for the synthesis of fatty acids [33], whereas the pathways for fatty acid synthesis and polyketide are highly interrelated
[20]. The SCDmanA mutant was already generated in this laboratory by the insertional inactivation of manA (SCO3025), which
encodes phosphomannose isomerase (PMI), exhibited a bld-like
phenotype, and failed to produce the antibiotics actinorhodin and
red tripyrolle undecylprodigiosin in liquid media [21]. Thus,
changes in the polyketide pool resulted in an increase in fatty acid
accumulation. Furthermore, when the mutant strain SCDmanA was
used in combination with RE, a production of 114 mg/L fatty acid
was obtained, which was 33% higher than the consortium of
SC:RE (86 mg/L) (Fig. 3). Different types of microbial consortia have
already been successfully used by different research groups for different purposes [16].
3.4. Biodiesel FAMEs composition and properties

The microbial consortia can perform more complicated tasks

and provide more changeable environments than monocultures,
which can affect the growth and production of metabolites [32].
The synthetic consortium of SC:RE was found to be the best consortia for enhanced fatty acid accumulation. To further increase the
fatty acid production, the manA mutant SCDmanA was analyzed,
revealing a 44% increase in biomass and 28% in fatty acid content,
respectively, compared with the wild-type SC. The increase in the

The accumulated fatty acids in the microbial consortia and individual microbes, were analyzed to determine the composition
(Figs. 4 and S1). The analysis was carried out for the short chain
fatty acids (SCFA, less than 6 carbons), medium chain fatty acids
(MCFA, 612 carbons), long chain fatty acids (LCFA, 1321 carbons)
and very long chain fatty acids (VLCFA, more than 22 carbons), as

Fig. 1. Biomass and fatty acid methyl esters proles of different bacterial strains.
Different microbes were used including S. coelicolor (SC), B. pumilus (BP), R. eutropha
(RE), and S. oneidensis (SO).

Fig. 3. Biomass and fatty acid methyl esters proles of selected consortia. An
examination was carried out on the consortia of SC:RE and SCDmanA:RE.

S.K. Bhatia et al. / Fuel 159 (2015) 189196

193

Fig. 4. GC/MS chromatograms of FAMEs produced using the microbial consortia of (a) SC:RE, and (b) SCDmanA:RE, along with RT and area. Deviation in RT is 0.05 min. Fatty
acid methyl esters (FAMEs) and RT: C13:0 (12.82), C12:0-3OH (13.95), C14:0-13M (15.94), C14:0-3OH (17.54), C15:0-14M (17.76), C16:1-n9 (18.13), C17:1 (19.87),
C18:2-n9,12 (21.45), C18:1-n9t (21.89).

well as the saturated and unsaturated fatty acid fractions (Table 2).
The fatty acids of SC and SCDmanA occurred in almost the same
ratio of MCFA:LCFA:VLCFA (5:94:1), whereas the increase in the
saturated and unsaturated fatty acids was 25% and 87%, respectively, for SCDmanA. In the case of synthetic consortia SC:RE and
SCDmanA:RE,
there
was
little
change
recorded
in
MCFA:LCFA:VLCFA (11.8:88.1:0.1 to 8.75:91.0:0.25), whereas saturated and unsaturated fatty acids increased by 30% and 37%,
respectively, for SCDmanA:RE. Finally, by using the synthetic consortium of SCDmanA:RE, a production of 114 mg/L fatty acids
was achieved, with 75% saturated fatty acid and 25% unsaturated
fatty acid content, which was 124% higher as compare to the pure
culture of SC (51 mg/L). The cetane number (CN) of biofuel is one of
its most important properties, as it determines the combustion
behavior of the biofuel, i.e., ignition delay time, which is the time
between the injection and ignition [34]. Biodiesel, with a higher
cetane number, have a shorter ignition time, and vice versa [35].
The CN value for the FAMEs produced by the synthetic consortium
of SCDmanA:RE was 65, which was higher than the other combinations (Table 3). According to the ASTM D6751-02 and EN14214
standards for biodiesel, the standard CN value must be a minimum
of 47 and 51, respectively [36]. The iodine value for the FAME of
SCDmanA:RE was 26, which is below the maximum value of
120 g/100 g, and represents the highest quality of this biodiesel.
Oxidative stability (OS) is also an important fuel property that is
related to the stability and performance of biodiesel [37]. The OS
value of SCDmanA:RE was 75.98, which is higher than that of the
FAMEs produced by the SC:RE consortium (31.41). The oxidative

stability was calculated based on the C18:2 and C18:3 contents

[38]. Stability is very important, as an unstable fuel can lead to
increased viscosity, as well as the formation of gums, sediment,
and other deposits, which affect the combustion behavior [39].
Other chemical and physical values were analyzed, including DU,
SV, LCDF, CFPP, CP, APE, BAPE, HHV, l, and q (summarized in
Table 3).
The lipid accumulation in microbes was studied by measuring
the Nile red uorescence intensity using uorescence spectroscopy. The uorescence intensity was measured for SCDmanA,
RE and SCDmanA:RE. The intensity of SCDmanA:RE was 1.5 higher
compared with SCDmanA, which shows the contribution of RE in
the enhancement of TAG accumulation (Fig. S2).
3.5. Consortia population dynamics and interaction
The organic acid prole was studied concerning the selected
strain SCDmanA and consortia SCDmanA:RE. The SCDmanA produce various organic acids (citrate and acetate), which leads to a
decrease in the pH of the media, down to 6.2, and slows down
the growth rate, while in consortia, RE utilize these organic acids
[40], and helps to maintain the pH of the medium around 7.4,
which resulted in an increase in the growth of SC. The population
dynamics of the microbial consortia SCDmanA:RE was studied
using a PLFA marker, as this method was previously used for the
community proling of microbes in decomposing conifers litter
[41]. The consortia SCDmanA:RE was cultured for 72 h, and samples were collected at an interval of 24 h. The biomass increased

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S.K. Bhatia et al. / Fuel 159 (2015) 189196

Table 2
FAMEs proles of different microbes and consortia.
FAME

RT

SC % (m/m)

RE % (m/m)

SCDmanA % (m/m)

SC:RE % (m/m)

SCDmanA:RE % (m/m)

C10:0
C10:0-2OH
C12:0-11M
C13:0
C12:0-3OH
C14:1
C14:0
C14:0-13M
C14:0-12M
C15:1
C15:0
C14:0-2OH
C14:0-3OH
C15:0-14M
C16:1-n9
C16:0 cyclo
C17:1
C16:0-15M
C16:0-2OH
C18:3-n6,9,12
C18:2-n9,12
C18:1-n9c
C18:1-n9t
C18:0 cyclo
C20:0
C22:6
C22:1
C23:0
C24:1
C24:0

6.34
8.84
12.17
12.82
13.88
14.58
14.91
15.88
16.09
16.38
16.66
16.92
17.47
17.72
18.09
19.46
19.82
19.82
20.57
21.06
21.52
21.67
21.83
24.12
27.10
29.92
30.96
32.70
33.51
33.51

5.038
0.843

24.77

1.433

0.436
34.20
2.281
20.07
0.191
1.424
1.424
1.081
1.497

0.105
2.686
0.472
0.134

0
0.572
0.444

0.145
0.131

4.97

5.47
0.475
0.501
0.183
0.412
0.488

0.284
13.05
43.56
2.751
0.576
0.576
2.610
3.728
12.27
0.476
3.511
0.130
2.152
0.135

1.176
0.338
0.624

0.285

0.345

4.808
3.197

21.45
0.106
4.207

0.460
24.08
4.160
29.70
1.246

1.904
0.531
0.169
1.632
0.136
0.819

0.289

0.124

0.598
0.862
10.364
2.824

21.532
0.101
7.694

0.464
26.08
2.559
22.04
0.799
0.010
1.257
0.183
1.129
2.992

0.800

0.557
.924
8.003
2.606

21.88

7.359

0.497
24.91
3.109
22.32
0.832
1.657
1.657
0.456
0.750
1.457

0.668

0.181

0.180

Table 3
Biodiesel properties of transesteried fatty acids of different consortia.
Parameter

SC

RE

SCDmanA

SC:RE

SCDmanA:RE

Units

SFA
MUFA
PUFA
DU
SV
IV
CN
LCSF
CFPP
CP
APE
BAPE
OS
HHV

75.23
23.27
1.50
26.27
225.82
26.90
64.42
0.37
15.31
3.73
5.70
3.27
0
36.56
1.03
0.82

35.07
47.90
16.13
80.16
216.29
80.35
53.46
3.94
4.10
1.89
36.66
24.59
9.97
38.32
1.16
0.85

68.36
29.84
1.80
33.44
233.07
33.19
62.25
0.72
14.21
3.08
3.73
2.55
68.14
38.01
1.08
0.86

73.08
22.80
4.12
24.78
235.92
24.48
63.93
0.22
15.79
3.82
2.78
0.99
31.41
37.70
1.04
0.85

74.81
22.99
2.20
25.89
227.46
25.51
64.56
0.47
15.00
3.45
3.57
1.81
75.98
36.76
1.03
0.83

% (m/m)
% (m/m)
% (m/m)

l
q

mg KOH/g oil
g I2/100 g
Min
% (m/m)
C
C

H
C
mm2/s
kg/m3

SC, S. coelicolor; RE, R. eutropha; SCDmanA, S. coelicolorDmanA; SC:RE, S. coelicolor: R. eutropha; SCDmanA:RE, S. coelicolorDmanA: R. eutropha; SFA, saturated fatty acids; MUFA,
monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; DU, degree of unsaturation; SV, saponication value; IV, iodine value; CN, cetane number; LCSF, long chain
saturated fatty; CFPP, cold lter plugging point; CP, cloud point; APE, allylic position equivalent; BAPE, bis-allylic position equivalent; OS, oxidation stability; HHV, higher
heating value; l, kinematic viscosity; q, density.

rapidly up to 48 h (2.4 g/L), after which very little increase in biomass was observed; the same results were obtained by a PLFA
analysis, as there was little change in the PLFA concentration after
48 h. The ratio of SC and RE in consortia SCDmanA:RE was calculated using a PLFA prole of pure culture (Fig. S3 a and b). The pure
culture of SC has 10Me16:0 as a PLFA marker (0.7 lg/g dcw),
whereas RE has a cy17:0 PLFA marker (0.62 lg/g dcw). At 24 h
the SC and RE ratio was 45:55, which changed to 52:47 at 48 h.
The nal ratio at 72 h was analyzed as 59:41 in the consortia
SCDmanA:RE (Fig. S4 ac).
Previously, fatty acid production has been reported, with the
use of various organisms Rhodococcus sp., Botryococcus braunii,
and various Streptomyces sp. (Table 4) [4244]. The algae species

are a good accumulator of fatty acid, but their cultivation time is

very long, compared with microorganisms [43]. Most of microbes
reported already utilize free sugars as a carbon source for oil accumulation, thus, there is a need to develop a system in which abundantly available biomass can be used as a carbon source [44]. Oil
accumulation in a pure culture of various Streptomyces sp. has
already been reported by many research groups and their productivity is close to the results of this study, but they have used only
free sugars; furthermore, there is no report on the properties of
oil produced by these systems. In this study, we have used a consortia approach that has its own advantages, i.e., the utilization
of starch as a carbon source by SC to make the process more economical, and coculture with RE, which does not compete with

195

S.K. Bhatia et al. / Fuel 159 (2015) 189196

Table 4
Fatty acids productivity comparisons with other reported systems.
Organism

Carbon source/medium

Culture time

Fatty acids (%)

Reference

Rhodococcus opacus PD 630

Botryococcus braunii
Rhodococcus opacus
Streptomyces albus
Streptomyces coelicolor 3(2)
Streptomyces griseus
Streptomyces lividans
S. coelicolorDmanA: R. eutropha

Cellobiose
Chu 13 media
Glucose/xylose
Glucose
Glucose
Glucose
Glucose
Starch

229 h
240 h
72 h
72 h
72 h
72 h
72 h
72 h

39.5
50.0
51.1
2.24
4.0
2.72
5.4
4.4

[42]
[43]
[44]
[11]
[11]
[11]
[11]
This study

the SC for carbon source; growing and adding more unsaturated

fatty acids by utilizing organic acids, the byproducts of fermentation, and thus improving the properties of the biodiesel that was
produced.
4. Conclusion
In this study, a semi-synthetic microbial consortia (semi-SMC)
approach was used to enhance the production of biodiesel with
an improved cetane number and oxidation stability. In the consortia of SCDmanA:RE, there is a synergistic interaction between the
two microbes, because the SC is providing organic acids as a carbon
source for RE, and in return RE maintains the pH of fermentation
media to promote the growth of SC, without any interference or
competition for the consumption of glucose. By using a microbial
consortium approach of this type, in this study we essentially
mimic nature, since similar phenomena can occur whereby different bacterial cells in a given ecosystem produce enzymes with distinct complementary specicities, which contribute to the overall
welfare of the community. These cell consortia may thus be more
amenable for consolidated bioprocessing strategies than a single
organism.
Acknowledgements
This paper was supported by Konkuk University in 2015.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.fuel.2015.06.084.
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