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Enhancing MFCs performance using novel artificial consortia.
Abstract
Microbial fuel cells (MFCs) hold great promise for the simultaneous treatment of wastewater and electricity production. However, the electricity recovery is currently poor, typically <10% of what is theoretically possible.
The influence of using cocultures as a way of improving substrate turnover rate and hence electricity produced was investigated using synthetic wastewater as a substrate. Cocultures used were combinations of Geobacter sulphurreducens, Clostridium beijerinckii and Saccharomyces cerevisae.
The best coculture combination was a mixture of Geobacter sulphurreducens, Clostridium beijerinckii and Saccharomyces cerevisiae giving a maximum power density of 80 mWm-2.
The best pure strain on power generation was C. beijerinckii and produced 74mWm-2. Maximum power produced by the pure strain of S. cerevisae and G. sulfurreducens were 38mWm-2 and 24mWm-2 respectively.
The best coculture on COD degradation were coculture of G. sulfurreducens and S. cerevisae; coculture of G. sulfurreducens and C. beijerinckii; coculture of S. cerevisae and C. beijerinckii giving 55% COD removal.
Sustained production of ethanol and butyric acid with concentration of 618mgml -1 and 6054mgml -1 respectively were achieved by the coculture of mixture of G. sulfurreducens, C. beijerinckii and S. cerevisae.
Sustained butyric acid of 4827mgml-1was also produced by the coculture of G. sulfurreducens and S. cerevisae.
Relative abundance analysis using RT-PCR showed dominance of G. sulfurreducens in the coculture of G. sulfurreducens and C. beijerinckii. The relative proportion of G. sulfurreducens to C. beijerinckii at the start and end were 36%:64% and 62%:38% respectively.
These results highlight the importance of cocultures in improving electricity recovery in microbial fuel cells. Further work will seek to investigate the influence of S. cerevisae and C. beijerinckii on the performance of MFCs using S. oneidensis in these conditions.
(Tesfaw and Assefa, 2014)(Tesfaw and Assefa, 2014)(Tesfaw and Assefa, 2014)(Tesfaw and Assefa, 2014)(Tesfaw and Assefa, 2014).
1.0 Introduction
World population and industrialization demands for energy/consumption are continuously increasing. The state of current energy consumption has almost depleted the fossils fuels and this has created a big threat to the future society. As a consequence, the cost of crude oil, coal, and natural gas is increasing from time to time. Current primary energy source is mostly provided by fossil fuels such as coal, petroleum, and natural gas that are non-renewable. The report by the Energy Information Administration (EIA) outlined that, world energy demand is growing about 0.6% in the Organization for Economic Cooperation and Development (OECD) member economies and 2.3% per year in the non-OECD economies from 2008 to 2035 (DOE/EIA-0484 (2011). The evaluated production years left for the proven reserves of oil, natural gas and coal are 35, 37, and 107 years, respectively. Awareness of global climate change and the uncertainty of fossil fuel have made several countries in the world, to respond to threats of energy security and global warming by diversifying their fuel sources to include renewable and alternative energy and developing clean energy technologies to replace the conventional ones. (Tesfaw and Assefa, 2014; Deepika et al., 2013 & Rahimnejad et al., 2014)
Generating energy from sustainable sources, such as biomass, is not only reliable and renewable but also helps reduce global carbon dioxide emissions. Hence, green energy is one of the options which can hopefully help us to achieve these needs. The discovery of biofuels as renewable energy sources has attracted attention these days. Bioethanol, biodiesel, and biogas are the dominant renewable energy among biofuels. A key method for generating renewable and alternative energy is through use of fuel cell technology. (Rahimnejad et al., 2014)
Microbial fuel cell (MFCs) is the emerging technology and hold great promise for the simultaneous treatment of wastewater and electricity production. However, the electricity recovery is currently poor, typically <10% of what is theoretically possible.
Several parameters have been reported to affect the performance of MFCs including microbial inoculums; the internal and external resistance of cells; chemical substrates; mass transfer areas; mechanism of electron transfer to the anode surface; solution ionic strength (shielding of charges in solution); electrode materials and the electrode spacing; absence or existence of proton exchange materials (PEM). PEM is an important parameter that can significantly reduce the performance of MFCs. The MFCs ion exchange capacity, conductivity and diffusion coefficient of MFCs can be reduced by PEM fouling (Xu et al., 2012). Inaddition to this, diffusion of oxygen from the cathode to
the anode through the PEM was reported (Du et al., 2007). Furthermore, resistance on proton transfer
from the anode to the cathode can significantly affect MFCs performance (Rabaey and Verstraete, 2005).
Process parameters critical to MFCs operation like cell metabolism; internal and external resistance; microbial electron transfer and cathode oxidation can be improved to enhance MFCs performance. Therefore, we hypothesized that the use of well-defined will enhance MFCs performance by increasing the rate of substrate utilization to electricity generation. In addition, complete anodic anaerobic condition will be enhanced by consumption of oxygen leakage to the anode chamber. Strains studied for co-culturing are S. cerevisae an electrogenic organism; G. sulfurreducens an electrogenic organism and C. beijerinckii a fermentative organism (Du et al., 2007).
2.0 Materials and method
2.1 Chemicals
Ethanol, butyric acid, acetic acid, sulphuric acid and glucose (purity ≥ 96%) where purchased from
Sigma Aldrich (UK). All chemicals were of analytical grade and were used without further
purification. Ficodox PlusTM mixed COD reagent was purchased from Fisher Scientific (UK).
2.2 Bacteria Strains, maintenance and cultures
S. cerevisae strain was obtained from the laboratory of life sciences from University of Westminster,
S. oneidensis strain 700550 was purchased from ATCC, G. sulfurreducens strain 51573 was obtained
from ATCC, C. beijerinckii strain ( ) was purchased from (). Cryopreserved stock cultures were
maintained at -800C. Strains were first cultured in Luria-Bertani broth medium (LB medium)
containing (per litre) 10g of tryptone, 5g of yeast extract and 5g of NaCl; later sub-cultured in
minimal salt medium (MSM) supplemented with 500mg/L glucose and modified LB medium
containing (g/L) tryptone – 10.0 and yeast extract – 5.0 as described in the section on operation of
BES - under 150 rpm at 300C for 48 hours. On the other hand, both strains were further grown in LB
medium supplemented with 15g l-1agar and plated for enumeration analysis before inoculation into the
MFC system.
2.3 Experimental design
The influence of S. cerevisae, S. oneidensis and C. beijerinckii on power generation from glucose
containing synthetic wastewater as energy source. They were checked for 10 days as pure isolates and
coculture under a strictly anaerobic-anodic conditions in two-chamber MFCs as described in section
2.4; at a volumetric ratio of 10% (V/V) of the anode working volume. The control was MFC with no
microorganism containing MSM medium supplemented with 500mg l-1 of glucose.
2.4 Bioelectrochemical system and operation
The H-type MFCs were constructed with two identical Duran bottles and were held together with an
external metal clip. The anode and cathode compartment were separated with a cation-exchange
membrane (CMI-7000, membranes International USA) to maintain an electro-neutrality in the fuel
cell system. Two rubber gaskets were used to ensure a seal. The electrodes were constructed from
carbon cloth. The cathode contained a Pt catalyst layer with a Pt loading of 0.5 mg cm-2. Both
electrodes had a projected surface area of 25 cm2. An external load of 1000 Ω was utilised in all
experiments and the potential across the resistor was recorded using the Picolog ADC-24 (Pico
Technology, U.K.) online data logging system.
The catholyte and anolyte solutions were buffered to pH 7.0 using 50mM phosphate buffer (21 mM -
NaH2PO4 and 29 mM – Na2HPO4) in all experiments. The anolyte minimal salts medium were
prepared by a little modification of Fernando et al., 2012 method. Briefly, consisted of (g l-1) NH4Cl –
0.46, KCl – 0.225, MgSO4.7H2O – 0.117, NaH2PO4 – 2.5, N2HPO4 -4.11, (NH4)2SO4 – 0.225 with
addition of vitamin mix and mineral solution of 1% (v/v) each respectively. The anolyte MSM was
supplemented with 500mg/L glucose and modified LB medium containing (g/L) tryptone – 10.0 and
yeast extract – 5.0. The catholyte was 50mM (pH 7) Phosphate buffer. The MFC components and all
media solutions for the experiment were sterilised by autoclaving. The experiments were conducted in
batch mode with a working volume of 200mL in each MFC compartment. During the start-up
operation the anode was seeded each with actively growing C. beijerinckii (18 x 108 CFU) or with G.
sulfurreducens (10 x 108) for the pure cultures while the coculture was with both inoculum
corresponding to 64% proportion of C. beijerinckii cells to 36% of G. sulfurreducens cells in the
coculture (5:5, 10% V/V of the total anolyte volume). The anolyte was purged with nitrogen gas for
10min through a 0.22 µm pore size diameter filter prior to inoculation. The cathoyte was actively
aerated at a flowrate of 100 mL air/min using an aquarium pump. All experiments were studied at
300C using a Stuart 160 incubator (Fisher Scientific, U.K.)
2.5 Analytical Procedure
2.5.1 COD removal
Chemical oxygen demand of the samples were determined using the closed reflux titrimetric method
as described in Environment Agency (UK) Standard method 5220D (Westwood, 2007). Briefly,
appropriate diluted 1 mL sample was used for each determination. The COD removal was calculated
by the following expression:
COD (mg L-1) = (Vb – Vs) *DF*M*4000
Where COD (mg L-1) represent the amount of oxygen consumed per litre of sample, Vb and Vs are
ferrous ammonium sulphate (FAS) titrant volumes for the blank and the sample respectively, DF is
the samples dilution factor and M is the molarity of FAS titrant.
The percentage COD removal was calculated as follows:
Percentage removal = (CODi - CODt) * 100 CODi
Where CODi and CODt are initial and final COD values of samples at the end of the investigation
respectively.
2.5.2 Detection of degradation product using Gas Chromatography
Degradation products of glucose were identified using Gas chromatography (GC) system. Briefly,
experimental samples (1.5 mL) for analysis were centrifuged at high gravitational force of 15,000 g
for 30 minutes using a centrifuge 5415 R. Thereafter, supernatant from each sample was transferred
into a 2 mL vial tube having a blue cap was run on a GC system. The GC was operated with the
following conditions: The mobile phase consisted of a carrier gas (helium) with a flow rate of
2mL/min; sample injection volume and middle injection nominal temperature was 1mL/min and
2600C respectively. The oven was initially set at 350C for 5 min and then ramped up to 1700C for the
subsequent 10 minutes. The three standard samples ethanol, acetic and butyric acid were detected at
2.959, 11.214 and 12.648 minutes respectively. The presence of degradation metabolites ethanol,
acetic acid and butyric acid were confirmed using the retention time (Rt) of the standard compounds.
2.5.3 Quantification of C. beijerinckii and S. oneidensis cells in the cultured medium
The unknown concentration of cells in the volume of cultured medium, used for inoculation of the
experimental system at the start of the investigation was determined by serially diluting the unknown
concentration by a known dilution factor of a tenfold series dilution. Cells for each strain were
aseptically plated on a LB agar medium and thereafter, incubated at 300C and 370C for S. oneidensis
and C. beijerinckii respectively for 24 hours to determine how many cells or colony forming units
(CFU) are present in the undiluted samples used for inoculation of the plate (plate count).
Calculations to determine the number of cells in the undiluted samples was determined by the
following expressions.
CFU/ml = Nc * D V
Where Nc is the number of cells counted on each plate, D is the dilution factor (10-7) and V is the
volume (mL) of aliquot of diluted cells added to each plate.
2.5.4 Relative abundance test by Real-PCR analysis
DNA extraction of samples were conducted using Bacterial Genomic DNA kit (Sigma Gen EluteTM).
Each plate included triplicate reactions per DNA sample and the appropriate set of standard. Relative
abundance of the bacteria coculture were obtained using real-time PCR (RT-PCR) by using specific
primers (Fierer et al., 2005) designed for proteobacteria, firmicutes and fungi. The primer specifically
target the 16S rRNA genes. The forward primer used for C. beijerinckii was Lgc353 with sequences:
GCA GTA GGG AAT CTT CCG and correponding reverse primer was Eub518: ATT ACC GCG
GCT GCT GG. Also, S. cerevisae was targeted using forward primer 5.8s and reverse primer 1TSif.
The reaction assay was conducted in strip tubes of 100 µL volume capacity each. Each 25 µL reaction
contained the following assay mixture: 12.5 µL of Absolute qPCR Master Mix (ABgene), 1.25 µL of
each primer (10 µM; Invitrogen), 25 µL bovine serum albumin (10 mg ml-1; Promega), 1.0 µL
SYBRGreen dye (16000- fold dilution in H2O), ROX dye (80-fold dilution in H2O; ABgene) 0.5 µL
nuclease free water and 5 µL of purified and in addition, 10 fold dilution series for each template
DNA (47.9 ng µL-1of C. beijerinckii, 57.1ng µL-1 of S. cerevisae and 42.5 ng µL-1of S. oneidensis)
were also assayed for analysis and for determination of the efficiency of the RT-PCR system. RT-
PCR amplification of samples were conducted using Quiagen Rotor-Gene system under the following
conditions: initial denaturation condition was 15 min at 950C, followed by 40 cycles of 950C for 1
min, annealing temperature at 600C for 30 s and 720C for 1 min according to the method of (Fierer et
al., 2005).
2.5.5 Electrochemical monitoring
Polarisation curve for measuring power density VS current density plots were constructed using a
range of external resistance ranging from 10Ω to 1 MΩ. The closed external circuit of the MFC
system for each test were opened to connect various external resistances when the system exhibited a
stable voltage across the initial 1000Ω external resistor. The current flowing through each external
load was calculated using the Ohm’s law (Fernando et al., 2012).
I = E/R
Where E is the potential across the resistor (mV), I is the current flowing through the load (mA) and R
is the external resistance (Ω).
The power generated was calculated with the following expression (Fernando et al., 2012)
P = E*I
Where P is the power produced, E is the potential (mV) and I is the current generated (mA).
The power density and current density values were calculated by normalising power and current
values to the projected surface area of the anodic electrode (25cm2).
Coulombic efficiency (CE) was calculated by integrating the measured current over time based on the
observed COD removal by using the criteria outlined in the literature (Zhao, et al. 2009).
3. Results
3.1 Assessment of electrochemical performance of the experimental MFCs system
The cocultures produced more power than the pure cultures as seen in Figure 2 and 3; table 1,
consistently with previous report (Read et al., 2010; Ren et al., 2007 and Qu et al., 2012). The highest overall power density 80mWm-2 among the studies were produced by the coculture of S. cerevisae, C. beijerinckii and G. sulfurreducens. The coculture of G. sulfurreducens and C. beijerinckii had the second highest power generation on the second day of 59mWm-2 but decrease to 54mWm-2 while C. beijerinckii was 74mWm-2 on the fourth day.
Looking at the highest power generated by the pure strains. C. beijerinckii produced 74mWm-2 on the second day; G. sulfurreducens produced 24mWm-2on the fourth day; S. cerevisae was 38mWm-2 on the second day. However, there was little significant difference on power produced on the second day by S. cerevisae and C. beijerinckii of 38mWm-2 and 39mWm-2 respectively.
On the second and fourth day, the coculture of C. beijerinckii and S. cerevisae, and coculture of G. sulfurreducens and S. cerevisae had poor performances generally. However, on the fourth day the highest power generated by the coculture of G. sulfurreducens and S. cerevisae was 15mWm-2. Whereas, no significant amount of power was generated by C. beijerinckii and S. cerevisae on both days.
The pattern of dual voltage generation associated with C. beijerinckii was produced in this study which was similar to previous investigation (Finch et al., 2011; Napoli et al., 2010) on glucose metabolism. The result on voltage generation is shown in Figure 1, the highest voltage across the study was 404mV produced by C. beijerinckii. The voltage produced by the coculture of C. beijerinckii, S. cerevisae and G. sulfurreducens, was 370mV and recorded the second highest voltage generated. Coculture of G. sulfurreducens and C. beijerinckii produced maximum voltage of 358mV while coculture of G. sulfurreducens and S. cerevisae produced 205mV.
There is little significant difference on voltage produced by the pure strain of G. sulfurreducens of 279mV and voltage produced by S. cerevisae of 273mV.
The least voltage across the test was generated by coculture of C.beijerinckii and S. cerevisae of 86mV. Hence, indicating the electrode was not used as electron acceptor and fermentation is the main process when potential is low (Rabaey and Verstraete, 2005).
0 2 4 6 8 10 12
0
50
100
150
200
250
300
350
400
450
G. sulfurreducens & S. cerevisae
S. cerevisae
G. sulf,S. cerevisae & C. beijerinckii
G. sulfurreducens & C. beijerinckii
G. sulfurreducens
C. beijerinckii & S. cerevisae
C. beijerinckii
Figure 1: Voltage generation by pure and defined mixed cultures of S. oneidensis and C. beijerinckii, S. cerevisiae and G. sulfureducens under closed circuit (1000Ω) condition for 10 days of investigation.
0 0.1 0.2 0.3 0.4 0.50
10
20
30
40
50
60
70
80
G. sulfurreducens & S. cerevisaeS. cerevisaeG. sulf,S. cerevisae & C. beijer-inckiiG. sulfurreducens & C. beijer-inckiiG. sulfurreducensC. beijerinckii & S. cerevisaeC. beijerinckii
Current density (Am-2)
Pow
er d
ensit
y (m
Wm
-2)
Figure 2: Power density versus Current density curve on Day 2
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80
10
20
30
40
50
60
70
80
90
G. sulfurreducens & S. cerevisaeS. cerevisaeG. sulf,S. cerevisae & C. bei-jerinckiiG. sulfurreducens & C. beijer-inckiiG. sulfurreducensC. beijerinckii & S. cerevisaeC. beijerinckii
Current density (Am-2)
Pow
er d
ensit
y (m
Wm
-2)
Figure 3: Power density versus Current density curve on Day 4 study
Table 1: Power density (mW m-2) and current density (A m-2) generation by Defined mixed cultures studies
Time (Days) Day 2 Day 4 Day 6 Day 8
Studies mW m-2 A m-2 mW m-2 A m-2 mW m-2 A m-2 mW m-2 A m-2 _____ _____ _____ _____ _____ _____ _____ _____S. oneidensis 54 0.14 94 0.35 85 0.33 41 0.13
S.onei + S. cere 75 0.24 65 0.23 52 0.17 42 0.24
G. sul + S. cere 8 0.04 14 0.08 20 0.06 21 0.09
S. cerevisiae 38 0.12 35 0.12 32 0.11 34 0.12
G.s + S. c+ C. be 69 0.23 80 0.25 12 0.07 6 0.03
G. sul + C. be 59 0.22 54 0.21 43 0.16 22 0.07
G. sulf 24 0.07 23 0.11 9 0.04 22 0.09
C. beij + S. cere 2 0.03 1 0.03 1 0.02 0.04 0.01
C. beije 39 0.18 74 0.39 20 0.09 13 0.1
3.2 Comparison of COD degradation and coulombic efficiency by the experimental studies
The results showed that the co-cultures are super-competitor as described by (Bernstein and Carlson, 2012). The results indicated enhanced resource utilization by the respective coculture and efficient depletion of the organic substrate than the pure cultures. Table 2 and Table 3 show respectively the amount of substrate consumed and product of metabolism. The result on substrate utilization by C. beijerinckii and G. sulfurreducens showed similarity of 32% on substrate degradation. There were similarities on substrate utilization by coculture of C. beijerinckii and S. cerevisae; G. sulfurreducens and S. cerevisae; G. sulfurreducens and C. beijerinckii of 55% metabolism by these studies.
The result suggested that COD degraded and the products of metabolism are majorly from glucose utilization. On the other hand, the amino acid component of the modified LB medium accounted for the remaining COD (%) not utilized by the experimental studies.
Amongst the study C. beijerinckii indicated 32% substrate utilization and coulombic efficiency of 13%. The high coulombic efficiency is consistent with the power produced by this system and indicated effective substrate utilization.
Although similar substrate utilization was recorded by the coculture as follows: coculture of C. beijerinckii and S. cerevisae with 55% COD degradation and 1 % coulombic efficiency; G. sulfurreducens and S. cerevisae with COD degradation of 55% and 4% coulombic efficiency; G.
sulfurreducens and C. beijerinckii with COD of 55% and coulombic efficiency of 6%, the result indicated significant increase on coulombic efficiency accordingly.
Focusing on coculture of S. cerevisae and G. sulfurreducens; C. beijerinckii and G. sulfurreducens. The result on the influence of both strains on G. sulfurreducens, indicated that C. beijerinckii significantly improve the coulombic efficiency by 2% and power generated by 38mWm -2 than S. cerevisae.
Although there was significant improvement on the substrate utilization by the performance of both cocultures when compared with the pure strain of G. sulfurreducens both cocultures showed reduction on coulombic efficiency than the pure culture of G. sulfurreducens. However, the coculture of G. sulfurreducens and C. beijerinckii produced more power than the pure culture of G. sulfurreducens by an increase of 35mWm-2. This relationship suggested enhancement of performance on power generation. Whereas the coculture of S. cerevisae with G. sulfurreducens showed reduction on power generation by 16mWm -2 and 2% on coulombic efficiency. Suggesting repression of G. sulfurreducens when coculture with S. cerevisae.
The MFCs with coculture of C. beijerinckii, S. cerevisae and C. beijerinckii indicated significant improvement on substrate degradation with an increase of 9% and power generation by 56mWm -2
than using pure strain of G. sulfurreducens. However, there were no significant differences on coulombic efficiency of 8% by both studies.
The performance of pure strain of S. cerevisae was higher than pure strain of G. sulfurreducens on power generation. The S. cerevisae recorded maximum power generation of 38mWm-2 whereas G. sulfurreducens was 24mWm-2. However, on the influence of C. beijerinckii when coculture with the electrochemically active strains, G. sulfurreducens had 59mWm-2 while S. cerevisae had 2mWm-2. Both coculture improved the substrate by 5% when compared with the pure strain of S. cerevisae. However, there was reduction in coulombic efficiency in both cocultures than the pure strain of S. cerevisae.
Table 2: Comparison of substrate degradation (%) and coulombic (%) efficiency between tests
Tests COD degradation (%) Coulombic efficiency (%)
C. beijerinckii + S. cerevisae 55 1
S. cerevisae 45 7
G. sulfurreducens 32 8
G. sulfurreducens + S. cerevisae 55 4
C. beijerinckii 32 13
G. sulfurreducens + C. beijerinckii 55 6
C. beijerinckii + S. cerevisae + G. sulfurreducens 41 8
3.3. Comparison of product of metabolism and power density across the tests
The result on the study of product of metabolism (Table 3) indicated that most of the ethanol and acetic acid produced on the second day were almost depleted on the tenth day of the study by the experimental systems that recorded significant amount of power generated. This observation indicated utilization of product of fermentation for power generation.
The result on the second day ethanol, acetic acid and butyric acid produced was totally depleted by coculture of G. sulfurreducens with beijerinckii; and pure strain of C. beijerinckii. This result showed similarities on utilization of product of fermentation.
However, ethanol produced by coculture of C. beijerinckii and S. cerevisae; pure strain of S. cerevisae showed similar utilization of product of metabolite. Comparatively these results indicated activities of C. beijerinckii and S. cerevisae in their coculture.
Concentration of acetic and butyric acid as high as 1963mgml-1 and 2979mgml-1 were produced respectively by S. cerevisae at the end of the investigation respectively.
The ethanol concentration of 618mgml-1 produced by the coculture of G. sulfurreducens, C. beijerinckii and S. cerevisae was retained and suggested the maximum concentration that can be generated by this system, while acetic acid was depleted from 3395mgml -1 to 8mgml-1 indicating cross-feeding occur in the system (Van Wey et al., 2014).
G. sulfurreducens produced the highest butyric acid of 6054mgml-1.
Similar observation on sustained production of butyric acid of 4827mgml -1was achieved by the coculture of G. sulfurreducens and S. cerevisae. The acetic produced was increased from the second to tenth day from 227mgml-1to 5080mgml-1 respectively indicating mutually-production of acetic acid.
G. sulfurreducens produced the highest overall acetic and butyric acid of 5482mgml -1and 6634mgml-1
respectively at the end of the study. While ethanol of 489mgml-1 was achieved.
Table 3. Comparison of product of metabolite from glucose degradation (500mgL-1) supplemented with modified LB medium, maximum power generation (Pmax) and fermentation product at the 2nd and 10th days of the investigation using Gas chromatography analysis.
Fermentation Product (mg/ml) Ethanol Acetic Butyric Acid Acid Acid Test 48h 240h 48h 240h 48h 240h
G. sulfurreducens 593 489 4105 5482 4153 6634
G. sulfurreducens + C. beijerinckii + S. cerevisae 618 618 3395 8 6054 6054
G. sulfurreducens + C. beijerinckii 773 ND 3894 ND 4555 ND
C. beijerinckii 952 0.73 5378 ND 5662 ND
C. beijerinckii + S. cerevisae 161 ND 4468 2912 4919 6371
S. cerevisae 2471 ND 5523 1963 3715 2979
G. sulfureducens + S. cerevisae 112 227 5080 4187 4827 4827
3.4 Relative abundances analysis of the cocultures
Environmental relationships, distribution and abundance was examined using RT-PCR. Generally the resource-based interactions showed co-existence relationship among the strains in the co-cultures.
The relative abundance analysis of the strains within the coculture indicated that C. beijerinckii reduced from 64% at the start to 38% at the end whereas G. sulfurreducens increased from 36% at the start to 62% at the end of the investigation. This result suggested repression of C. beijerinckii by G. sulfurreducens in the coculture. The increase in the performance of the coculture can be due to increase in the proportion of G. sulfurreducens.
Although the amount of inoculated S. cerevisae cells was not enumerated at the start of the investigation. However, looking at the result on the coculture of G. sulfurreducens with S. cerevisae, the result suggested G. sulfurreducens was repressed by S. cerevisae. Indicating relative abundance ratio of 15% and 85% for G. sulfurreducens and S. cerevisae respectively. The result can be attributed to the poor maximum power generated of 21mWm -2 by this system. Although there was consistent significant increase on the power generated by this system. This can be due to increase in the proportion of G. sulfurreducens cells in the system.
The coculture of C. beijerinckii and S. cerevisae ratio of 71% and 29% respectively suggested repression of S. cerevisae. This can be related to the poor performance recorded by this system.
Table 3. Comparison of relative abundance of the two target organisms in the coculture tests
test % composition
G. sulfurreducens C. beijerinckii S. oneidensis S. cerevisae
G. sulfurreducens, C. beijerinckii 62 38 NA NA
G. sulfurreducens, S. cerevisae 15 85 NA NA
C. beijerinckii, S. cerevisae NA 71 NA 29
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.50
100
200
300
400
500
600
700
800
G. sulfurreducens & S. cerevisaeS. cerevisaeG. sulf,S. cerevisae & C. beijer-inckiiG. sulfurreducens & C. beijerinckiiG. sulfurreducensC. beijerinckii & S. cerevisaeC. beijerinckii
Current density (Am-2)
Volta
ge (m
V)
Figure 4: Polarisation curves on day 2 of data produced by the experimental factors.
0 2 4 6 8 10 120
100
200
300
400
500
600
700
G. sulfurreducens & S. cerevisae
S. cerevisae
G. sulf,S. cerevisae & C. beijer-inckii
G. sulfurreducens & C. beijer-inckii
G. sulfurreducens
C. beijerinckii & S. cerevisae
C. beijerinckii
Current density (Am-2)
Volta
ge (m
V)
Figure 5: Polarisation curves on day 4 of data produced by the experimental factors.
0 0.1 0.2 0.3 0.4 0.5 0.6 0.70
100
200
300
400
500
600
700
G. sulfurreducens & S. cerevisaeS. cerevisaeG. sulf,S. cerevisae & C. beijerinckiiG. sulfurreducens & C. beijerinckiiG. sulfurreducensC. beijerinckii & S. cerevisaeC. beijerinckiiS. oneidensis & S. cerevisaeS. oneidensis
Figure 6: Polarisation curves on day 6 of data produced by the experimental factors.
Table 3. Comparison of product of metabolite from glucose degradation (500mgL-1) supplemented with modified LB medium, maximum power generation (Pmax) and fermentation product at the 2nd and 10th days of the investigation using Gas chromatography analysis.
Fermentation Product (mg/ml) Ethanol Acetic Butyric Acid Acid Acid Test 48h 240h 48h 240h 48h 240h
G. sulfurreducens 593 489 4105 5482 4153 6634
G. sulfurreducens + C. beijerinckii + S. cerevisae 618 618 3395 8 6054 6054
G. sulfurreducens + C. beijerinckii 773 ND 3894 ND 4555 ND
S. oneidensis 117 ND 6632 ND 4109 ND
C. beijerinckii 952 0.73 5378 ND 5662 ND
C. beijerinckii + S. oneidensis 161 ND 4468 2912 4919 6371
S. oneidensii + S. cerevisae 78 20 5728 6360 3882 5477
S. cerevisae 2471 ND 5523 1963 3715 2979
G. sulfureducens + S. cerevisae 112 227 5080 4187 4827 4827
4.0 Discussion
An approach to enhance MFCs performance by maximizing substrate turn-over rate since to electricity generation was investigated in this study. The choice of selection of strains were based on the knowledge of microorganism rarely live in isolation but are often found in consortium (Van Wey et al., 2014); G. sulfurreducens function as an electrogenic obligate anaerobic organism; S. cerevisae function as a facultative- electrochemically active organism and can consume oxygen intrusion to the anode chamber of the MFCs and C. beijerinckii function as a fermentative organism.
The energy containing compound present in waste water can be metabolized by microorganisms as biocatalyst resulting in electron gain. The by-product of metabolism, the electron (e-) and protons (H+) released result in the development of bio-potential which facilitates bioelectricity generation (Mohan et al., 2009)
The well-defined coculture combination of G. sulfurreducens, C. beijerinckii and S. cerevisae outperform the pure cultures. The interactions of individual microbes in the coculture of G. sulfurreducens, C. beijerinckii and S. cerevisae showed enhanced productivity and stability on power generation and product of metabolism. The COD % degraded showed that the coculture had enhanced metabolic functionality.
The high COD (%) not utilized suggested the presence of amino acids compounds and accounted for most of these values. The result on utilization of product of fermentation supported glucose degradation and utilization by these experimental systems.
The poor performance of MFCs utilizing pure culture of G. sulfurreducens suggests inhibition by oxygen leakage into the system. S. cerevisae produced more power maybe due capability of utilising oxygen leakage into the anodic chamber of the system for effective MFCs performance.
This work further supported the observation that C. beijerinckii has the capability of producing electricity generation in MFCs and performed the best amongst the pure cultures on electricity production.
Implications from this findings suggests the possibility of enhancing MFCs performance simultaneously on bioremediation and power generation by using coculture of well-defined strains of microorganism.
Conclusion
Briefly, this work indicates that cleverly-defined coculture enabled more electrical power generation and wastewater treatment than the monoculture systems. The result suggests that oxygen sensitive G. sulfurreducens should consortially be applied with an oxygen scavenging microbes when applied in MFCs operation. The influence of C. beijerinckii and S. cerevisae on MFCs performance utilizing S. oneidensis is suggested for future investigation
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