ORIGINAL PAPER

Symbiosis of photosynthetic microorganisms with non-photosynthetic ones for the conversion of cellulosic massinto electrical energy and pigments

R. Navanietha Krishnaraj • Sheela Berchmans •

Parimal Pal

Received: 12 March 2014 / Accepted: 31 May 2014

� Springer Science+Business Media Dordrecht 2014

Abstract In this article, we report a three-compart-

ment microbial fuel cell (MFC) system for the

simultaneous degradation of cellulose and production

of natural pigments such as phycoerythrin and phyco-

cyanin along with bioelectricity generation. Oscilla-

toria annae, a freshwater cyanobacterium, was used

for the conversion of cellulose to reducing sugars,

which were fed as a substrate to a coculture of

Acetobacter aceti and Gluconobacter roseus for

current generation in a three-compartment MFC.

Carbon felt modified with a composite film containing

chitosan and sodium alginate served as the MFC

anode. The cellulose-fed three-compartment MFC

produced a maximum power output of 6.62 W m-3 at

17.55 A m-3.

Keywords Cellulose � Pigments � Bioelectricity �Microbial fuel cell

Introduction

Microbial fuel cells (MFCs) represent green tech-

nology bioelectrochemical systems in which the

electrigens oxidize the substrate in anaerobic condi-

tions and transfer the electrons to the anode (Logan

2009). MFCs have several operational and functional

advantages. However, the low power output and cost

factor remain its weaknesses (Dewan et al. 2010).

MFCs are likely to become viable if the economics

of running a sustainable fuel cell can be sorted out.

Further synthesis of novel chemical products along

with electricity production would be advantageous.

The use of a cheap and abundant biomass may help

to reduce the operational cost of the MFCs (Logan

and Rabaey 2012). Cellulosic biomass seems to be a

cheap and attractive carbon-neutral renewable

resource for use as a substrate in MFCs because of

its abundance. Use of cellulosic wastes in MFCs

could help to solve the current global energy crisis

and remediate the huge volume of cellulosic waste

(Rezaei et al. 2009).

The use of cellulosic biomass in any bioprocess

operation is mainly hindered by the recalcitrant

nature of cellulose, which makes the use of these

materials as substrates directly in MFC a challenge.

Hence, pretreatment is necessary for breaking the

structural and chemical complexity of the biomass

(Mosier et al. 2005). Cellulose can be hydrolytically

broken down into glucose by biological, physical or

chemical methods. Physical methods such as ball

Electronic supplementary material The online version ofthis article (doi:10.1007/s10570-014-0319-y) contains supple-mentary material, which is available to authorized users.

R. Navanietha Krishnaraj � S. Berchmans (&)

CSIR-Central Electrochemical Research Institute,

Karaikudi 630006, Tamil Nadu, India

e-mail: sheelaberchmans@yahoo.com

P. Pal

National Institute of Technology,

Durgapur 713209, West Bengal, India

123

Cellulose

DOI 10.1007/s10570-014-0319-y

milling and thermal methods are not suitable for real-

time applications. Chemical methods make use of

strong acids and solvents for the hydrolysis of

cellulose. Problems such as acid recovery, the

complicated recycling process and cost factors make

these methods unsuitable for commercial applica-

tions. If the acid is volatile, vacuum stripping

methods are also need to be practiced (Hendriks

and Zeeman 2009; Ladisch et al. 1978). Enzymatic

hydrolysis has several disadvantages, such as the

narrow temperature and pH range of the enzymes,

high costs, slow hydrolysis rate and need for

sophisticated facilities and huge reactors (Orozco

et al. 2007). Microbial hydrolysis offers many

advantages over other methods because of the higher

yields, low energy requirements, mild operating

conditions and ecofriendly nature. Certain species

of cyanobacteria possess excellent lignolytic and

cellulolytic activities. Metagenomic analysis of the

wood-decomposing microbial community revealed

the presence of a few cyanobacterial species in

association with other cellulolytic and lignolytic

organisms (Van der Lelie et al. 2012). As these

organisms have simple growth requirements, cultur-

ing the cyanobacteria in large amounts is less

expensive and can be used effectively for degrading

celluloses (Kumar Saha et al. 2003; Gupta et al.

2011). Acetic acid bacteria are ideal options for

bioelectricity generation because of their good oxi-

dizing ability for a wide range of substrates (Navan-

ietha Krishnaraj et al. 2013; Karthikeyan et al. 2009;

Navanietha Krishnaraj et al. 2014). The metal-

reducing characteristics of Gluconobacter roseus

have also been documented in the literature (Navan-

ietha Krishnaraj and Sheela 2013b). One report in the

literature describes the use of Enterobacter cloacae

for current generation along with degradation of

cellulose (Rezaei et al. 2009; Ren et al. 2007).

In this work, cellulolytic activity is provided by

cyanobacteria, and electrogenic activity is pro-

vided by the coculture of G. roseus and Aceto-

bacter aceti. Herein, we have also demonstrated

for the first time the symbiotic effect of a

photosynthetic organism and a coculture of non-

photosynthetic organisms for the effective degra-

dation of cellulose along with current generation

and the production of natural pigments, illustrating

the scope of using abundant cellulosic biomass for

energy production.

Experimental

MFC construction

A three-chambered MFC was constructed with a

Perspex sheet. The dimensions of the first chamber

were 5 cm 9 3 cm 9 4.5 cm, and the second and

third chambers had the same dimensions of

3 cm 9 3 cm 9 4.5 cm. The first and second cham-

bers were separated by a cellophane membrane. The

second and the third chambers were connected by a

2 cm 9 2-cm proton exchange membrane (Nafion

115). A. aceti (NCIM No. 2116) and G. roseus (NCIM

no. 2049) was procured from NCL, Pune, India.

Oscillatoria annae was procured from the National

Facility for Marine Cyanobacteria, Tiruchirappalli,

India. The first chamber contained cyanobacteria (1 g

wet weight) in BG-11 media (composition of BG-11

media is shown in the supplementary information); the

second chamber contained the anode with the biofilm

and the anolyte (phosphate buffer), and the third

chamber contained the carbon felt cathode and the

catholyte (3.3 g of potassium ferricyanide in 100 ml

of buffer).

Modification of the anode

The electrode modification procedure has already

been documented in the literature (Navanietha Krishn-

araj et al. 2013; Navanietha Krishnaraj and Sheela

2013a). Bare carbon felt (3.18 mm thick, procured

from Alfa Aesar) of 2 cm 9 2-cm dimensions was

modified by electrodepositing 1 % chitosan in 0.1 M

acetic acid at -10 V. The chitosan-deposited elec-

trode was left undisturbed until it became dry. Then,

an over layer of sodium alginate was formed by dip

coating in a 2 % sodium alginate solution and allowed

to dry further. Thus, the formed chitosan-sodium

alginate-coated felt was kept in 2 % CaCl2 overnight

for the formation of calcium alginate. Unmodified

carbon felt was used as a control for comparison.

Formation of the biofilm

The bare and the modified electrodes were kept in

phosphate buffer containing glucose (0.2 g/30 ml of

buffer) and a coculture of A. aceti and G. roseus (wet

weight of 0.1 g A. aceti and 0.1 g G. roseus) for

biofilm formation in stirred conditions until a stable

Cellulose

123

negative potential had been reached. Aneorobic con-

ditions were maintained during biofilm formation.

MFC operation and performance

The first chamber contained the cyanobacteria in BG

11 media. The anode compartment (second compart-

ment) of the three-chamber MFC was completely

deaerated with nitrogen gas. Cellulose acetate (0.03 g)

was used as the substrate. Modified carbon felt

(2.5 cm 9 2.5 cm) was used as the anode, and bare

carbon felt was used as the cathode. A data logger

(Agilent acquisition 34970A data acquisition) was

used to measure the voltage difference between the

anode and cathode across the fixed external resistance

at 5-min intervals. The data were collected automat-

ically by a data acquisition program and personal

computer. Then, the current (I) was calculated using

the formula I = V/R, where V is voltage and R the

applied resistance. Current density, j (A m-3), was

calculated using the formula j = I/v; power density,

P (W m-3), was calculated using the formula P = IV/

v, where v is the volume of the anolyte (15 ml).

Polarization studies were carried out in MFCs by

applying variable resistances between 10,000 and 100

X, and the final steady-state voltage was recorded for

each applied resistance as reported previously (Kart-

hikeyan and Sheela 2012). Ohmic resistance was

calculated from the slope of the polarization curve at

the linear (ohmic) region (Fan et al. 2008). A duplicate

experiment with an unmodified carbon felt anode was

carried out in an identical MFC for comparison. MFCs

with bare and modified electrodes were discharged

under a constant load, and their coulombic efficiencies

were calculated based on the following formula:

n ¼ Qobs=Qtheor � 100

where Qobs = the current gained under constant load

(C), and Qtheor = quantity of current expected from

the glucose consumption under constant load (C) (You

et al. 2006). Catalytic oxidation of fuel by the

microorganisms was analyzed by measuring COD

changes every 24 h (Logan et al. 2006). O. annae was

used for hydrolyzing the cellulose, and the kinetics of

hydrolysis of cellulose in the three-chambered MFC

were calculated by estimating the reducing sugar

levels (Miller 1959). After the completion of the

experiment, 5 ml of cyanobacterial culture from the

first chamber was centrifuged at 8,000 rpm. The

pigments were extracted, and the concentration of

the phycobiliproteins was calculated from the absorp-

tion spectrum of the supernatant in the wavelength

region of 250–700 nm. The C-phycocyanin, allophyc-

ocyanin and the C-phycoerythrin were quantified as

described elsewhere (Bennett and Bogorad 1973). The

acetone extract of the cyanobacterial biomass was

used to measure chlorophyll-a (663 nm) and carote-

noids (450 nm)(Mishra et al. 2012; McKinney 1941).

Electrochemical characterization of biofilm

Carbon felts of smaller dimensions (1 cm 9 0.4 cm)

were used for electrochemical characterization. A

brass rod was used for electrical contact. The electrode

was modified as described in ‘‘Modification of the

anode.’’ Bare and modified carbon felt electrodes in

the presence of biofilm were examined by cyclic

voltammetry in phosphate buffer in the potential

region from -1 to ?1.2 V at 50 mV s-1. A normal

calomel electrode (NCE) and Pt foil were used as the

reference electrode and the counter electrode,

respectively.

SEM analysis of microbial growth

The biofilm of the coculture formed over the bare and

modified felt was characterized by SEM analysis. A

piece of the bare and modified felt with biofilm felt

was carefully cut to 1 cm 9 1-cm dimensions in

aseptic conditions. The felt was gold sputter coated,

and the analyses of the biofilms were carried out using

the SEM Hitachi model-S-3000H unit.

Results and discussion

Modification of the electrode

Modification of the electrode was clearly investigated

using cyclic voltammetry analysis. The change in the

cyclic voltamogram indicates the effect of function-

alization of the carbon felt electrode with chitosan

and alginate. The cyclic voltamogram of bare carbon

felt, carbon felt modified with chitosan and carbon

felt modified with chitosan and sodium alginate

shows the increase in the surface area of the

electrode on modifying the electrode with chitosan

and alginate. The cyclic voltammograms of bare

Cellulose

123

carbon felt, carbon felt modified with chitosan and

carbon felt modified with chitosan and sodium

alginate are shown in Fig. 1.

Hydrolysis of cellulose, polarization studies

and coulombic efficiency

Oscillatoria annae hydrolyzed cellulose to reducing

sugars such as cellobiose and glucose. Figure 2a

shows the reducing sugar levels in the MFC. Forma-

tion of reducing sugars can be due to the release of

endogluconases of O. annae. The hydrolysis rate was

initially slow during the operation, and it increases

gradually to the maximum and then decreases.

Figure 2b, c shows the polarization curves for the

MFCs constructed with bare carbon felt and modified

carbon felt anodes, respectively. Polarization studies

with a bare carbon felt anode produced a maximum

power density of 4.55 W m-3 at 14.22 A m-3. The

power density of the MFC with the chitosan-alginate

modified anode was found to be 6.62 W m-3 at

17.55 A m-3. The power density and current density

of the MFC with the modified anode increased by 45.5

and 23.4 %, respectively, when compared with the

MFC with an unmodified anode. The power density of

the MFC was mainly affected by ohmic resistance.

The ohmic resistance of the three-chambered MFC

with a bare and modified anode was found from the

slope of the linear region as the entire polarization

curve is not strictly linear. In the bare anode MFC, the

ohmic resistance was found to be 975.5 X between the

potential regions from 0.064 to 0.32 V. The slope

value in the linear region in the polarization curve of

the MFC with the modified anode was found to be

617.6 X between 0.287 and 0.08 V. The modification

of the anode decreases the ohmic resistance by

57.9 %. Coulombic efficiency, which determines the

scaling up and application of MFC of the device for

applications, was found to be 11.65 and 27.64 %,

respectively, for MFCs with unmodified and modified

anodes.

-1.0 -0.5 0.0 0.5 1.0 1.5-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3Bare carbon felt Carbon felt modified with chitosan Carbon felt modified with

Chitosan-Alginate

Cu

rren

t ( m

A)

Potential (V vs NCE)

Fig. 1 Cyclic voltammograms of bare carbon felt, carbon felt

modified with chitosan and carbon felt modified with chitosan

and sodium alginate

0 5 10 15 20 25 300

1

2

3

4

5

Current density/Am-3

Po

wer

den

sity

(W/m

3)

0.0

0.1

0.2

0.3

0.4

Vo

ltage/V

B

0 5 10 15 20 25 30 350

1

2

3

4

5

6

7

Current density (A/m )3

Po

wer

den

sity

(W/m

)

3

C

0.0

0.1

0.2

0.3

0.4

0.5

Vo

ltage/V

A

Fig. 2 a Cyanobacteria-aided hydrolysis of cellulose in a three-

chambered MFC. Polarization characteristics of the three-

chambered MFC with b a bare carbon felt anode and c chitosan-

sodium alginate composite modified anode

Cellulose

123

Bioelectrocatalysis of the biofilms

The electroactive properties of the biofilms were

analyzed by cyclic voltammetry. The redox activity of

the biofilm is indicated by the presence of quasi

reversible peaks appearing at 0.1 and -0.3 V. The

peaks corresponding to the biofilms formed on both

bare and modified electrodes indicates the presence of

electroactive redox species in the biofilm. Electrocat-

alytic activity of the biofilm was evaluated by adding

the reducing sugar solution formed during the hydro-

lysis of cellulose by cyanobacteria. The sample

containing 20 mM of reducing sugar (quantified using

a spectrophotometer) was added as the fuel. Figure 3a,

b shows the bioelectrocatalytic properties of the

biofilm toward the oxidation of reducing sugars

derived from cellulose. For an addition of 20 mM of

the substrate, the current rises to 0.653 from 0.598 mA

in the case of unmodified anode. On second addition of

the substrate, the current further increases to

0.708 mA. In the case of the modified anode, the

addition of 20 mM of the reducing sugar increased the

current from 0.555 to 0.901 mA. Further addition of

20 mM of reducing sugar increased it to 1.190 mA.

This significant increase in the current by 82 % in the

case of the modified electrode clearly indicates the

enhanced efficiency and suitability of the chitosan-

sodium alginate anodes for MFC applications.

Fuel consumption

The fuel consumption was analyzed by measuring the

COD levels in both the first and second chamber every

24 h over a 20-day period. The average rate of fuel

consumption on the 7th day was found to be 44 and

65 % in an MFC with bare and modified anodes,

respectively, in the first chamber (Fig. 4a). This

increased COD change was due to the utilization of

fuel in the second chamber. Then, on the 8th day,

0.01 g of cellulose acetate was added. The percentage

COD removal in the second cycle was found to be 62

and 69 % in the first chamber of an MFC with a bare

and modified anode, respectively. On the 15th day,

0.01 g of cellulose acetate was added again. The

average COD removal rate in the first chamber of the

MFC with bare and modified anodes was found to be

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5 Biofilm

1st Addition of 20mM glucose

2nd addition of glucose

(O. annae hydrolysed cellulose)

Cu

rren

t (m

A)

Potential (V vs NCE)

0.598mA0.653mA0.708mA

A

-1.0 -0.5 0.0 0.5 1.0

-1.0 -0.5 0.0 0.5 1.0-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0 Biofilm

1st addition-20mM Glucose

(O.annae hydrolysed cellulose) 2 nd addition-20mM Glucose

Cu

rren

t (m

A)

Potential (V vs NCE)

0.555mA0.901mA1.190mA

B

Fig. 3 Cyclic voltammograms showing the effect of the

addition of reducing sugar derived from cellulose on the

biofilms: a carbon felt anode and b chitosan-sodium alginate

composite modified anode

Fig. 4 COD levels in the MFC with a bare and modified anode

a in the first chamber and b in the second chamber (anode

compartment)

Cellulose

123

48 and 73 %, respectively. A similar study in the COD

levels was performed for the anolyte in the second

chamber and is shown in Fig. 4b. The COD levels

were initially very low in the second chamber of both

bare and modified anode MFCs. It slowly increased

and reached the maximum on the 3rd day after the

addition of substrate in the first chamber. Then, the

COD levels decreased on the 6th and 7th days. Then,

after the addition of substrate for the second cycle, the

COD levels increased after 2 days and decreased on

the 13th and 14th days, and this trend continued in the

third cycle. A similar pattern was observed in MFCs

with modified and unmodified anodes.

Production of cyanobacterial pigments

Different pigments were produced from the cyanobac-

terial biomass produced in the MFC. The biomass

contained 18.46783 mg l-1 of C-phycoerythrin,

13.09972 mg l-1 of allophycocyanin and

20.38901 mg l-1 of C-phycoerythrin. Chlorophyll-

a and carotenoid contents in the biomass were found

to be 0.376788 and 0.453 mg l-1 respectively. The

oxidation reactions in the second chamber did not affect

the production of pigments in the MFCs.

Morphological characterization of biofilms on bare

and modified electrodes

The morphological characterization of the biofilms on

bare and modified carbon felt was performed using

SEM. Figure 5a represents the biofilm on the bare

carbon felt anode. Figure 5b represents the SEM

image of the biofilms formed on the Chit–Alg/carbon

felt anode. It clearly shows the biocompatibility of the

chitosan-alginate composite enhances the biofilm

formation on the electrode.

Conclusion

The synergistic effect of two different types of

microorganisms has been capitalized for effective

degradation of cellulose resulting in the production of

pigments and electricity. The pigments could be of

substantial use in pharmaceuticals as antioxidants, as

food colorants, in cosmetics, in fluorescent labeling

and as a sensitizer for dye-sensitized solar cells. This

investigation brings out the scope of the effective

utilization of naturally abundant cellulosic biomass.

Acknowledgments The author, R. Navanietha Krishnaraj,

acknowledges CSIR, New Delhi, for the junior research

fellowship.

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