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Simultaneous organic carbon, nutrients removal and energy production ina photomicrobial fuel cell (PFC)†

Yifeng Zhang, Jafar Safaa Noori and Irini Angelidaki*

Received 6th July 2011, Accepted 17th August 2011

DOI: 10.1039/c1ee02089g

A sediment-type photomicrobial fuel cell (PFC), based on the synergistic interaction between

microalgae (Chlorella vulgaris) and electrochemically active bacteria, was developed to remove carbon

and nutrients from wastewater, and produce electricity and algal biomass simultaneously. Under

illumination, a stable power density of 68 � 5 mW m�2 and a biomass of 0.56 � 0.02 g L�1 were

generated at an initial algae concentration of 3.5 g L�1. Accordingly, the removal efficiency of organic

carbon, nitrogen and phosphorus was 99.6%, 87.6% and 69.8%, respectively. Mass balance analysis

suggested the main removal mechanism of nitrogen and phosphorus was the algae biomass uptake

(75% and 93%, respectively), while the nitrification and denitrification process contributed to a part of

nitrogen removal (22%). In addition, the effect of illumination period on the performance of PFC was

investigated. Except notable fluctuation of power generation, carbon and nutrients removal was not

significantly affected after changing the light/dark photoperiod from 24 h/0 h to 10 h/14 h. This work

represents the first successful attempt to develop an effective bacteria–algae coupled system, capable for

extracting energy and removing carbon, nitrogen and phosphorus from wastewater in one-step.

1. Introduction

Urban wastewater contains organic carbon and nutrients, which

can cause eutrophication and deterioration of the natural water

quality. These substances need to be removed or captured, before

Department of Environmental Engineering, Technical University ofDenmark, DK-2800 Lyngby, Denmark. E-mail: iria@env.dtu.dk; Fax:+45 45932850; Tel: +45 45251429

† Electronic supplementary information (ESI) available: Algaeconcentration versus optical density (OD) at 658 nm (Fig. S1). DO andpH change with time (Fig. S2). Sediment characteristics of BagsvaerdLake DGGE (Table S1) and 16S rDNA library analysis (Table S2). SeeDOI: 10.1039/c1ee02089g

Broader context

Microbial fuel cells (MFCs) are a promising technology for simul

Anodic conversion of organic carbon into electricity is a relatively ea

too expensive since costly membranes and mechanical aeration a

removal in MFCs. Fortunately, microalgae technology for wastewa

a novel, efficient and cost-effective solution for nutrients removal in

cell (PFC), based on the cooperation between microalgae and elec

nation, a stable power density of 68� 5 mWm�2 and a biomass of 0

of 3.5 g L�1. Accordingly, the removal efficiency of organic carb

respectively. The performance was further improved by optimizatio

that the PFC system offers a straightforward, effective and cost-eff

simultaneously, opening an entirely and environmentally friendly w

4340 | Energy Environ. Sci., 2011, 4, 4340–4346

being reused or returned to the environment. While high chem-

ical oxygen demand (COD) removal has been easily achieved in

many wastewater treatment systems, the nutrients removal is

a more complex and costly process which involves several steps

and technologies.1–3 Advanced technologies for both carbon and

nutrients removal that minimize environmental impacts and

recovery energy are now given high priority.

Recently, microbial fuel cells (MFCs) have drawn much

attention as a new approach to treat wastewater, replace energy

intensive wastewater treatment processes, and produce clean

electric energy or valuable products.4–8 In power-generating

MFCs, electrochemically active bacteria transfer electrons

produced during oxidation of organic matter to the anode; while

taneous wastewater treatment and electric energy production.

sy process inMFCs. However, nitrogen removal inMFCs is still

re required. Moreover, there is still no report of phosphorus

ter nutrients treatment has been recently developed, providing

MFCs. Here, an innovative sediment-type photomicrobial fuel

trochemically active bacteria, has been reported. Under illumi-

.56� 0.02 g L�1 were generated at an initial algae concentration

on, nitrogen and phosphorus was 99.6%, 87.6% and 69.8%,

n of the algae concentration and C/N ratio. This demonstrates

ective route to remove C, N, P and recover energy and biomass

astewater treatment concept.

This journal is ª The Royal Society of Chemistry 2011

Fig. 1 Schematic illustration of the functional principles of the PFC (a)

and photoimage of the reactor (b).

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oxygen is reduced by accepting electrons from the cathode.5,9,10

Electricity can be generated from various biodegradable organic

materials in MFCs, including carbohydrates,11,12 low molecular

weight organic acids,13 proteins,14wastes and waste streams, such

as dairy manure,15 domestic wastewaters,5 and food process

wastewater.16 Anodic conversion of organic carbon into elec-

tricity is a relatively easy process in MFCs and has already been

widely proven in the literature mentioned above. However,

nutrients removal in MFCs, on the other hand, has only received

a little attention so far.17–19 In recent studies, simultaneous

organic matter, nitrogen removal, and power production were

achieved in two-chamber MFC reactors where nitrification was

accomplished by a specific aeration.18,19 Even though electric

energy and nitrogen removal can be achieved by MFCs, the

technology is still too expensive; the employed membranes are

costly, and a considerable amount of energy is consumed for

nitrification in terms of mechanical aeration. Furthermore,

MFCs have little capacity to remove phosphorus, and there is

no report of phosphorus removal by MFC technology available

so far. Fortunately, the emergence of microalgae technology

may bring a good complement to MFCs. It has been known for

several years that microalgae can assimilate nitrogen and

phosphorus into their biomass as well as carbon dioxide for

photosynthesis and produce oxygen, while the biomass can be

further used for the production of valuable products (e.g.,

biodiesel, fertilizer).20,21 However, microalgae have less capacity

for organic carbon removal through photosynthesis, have to

cooperate with aerobic bacteria to degrade organic carbons,

and there is no energy recovery from this process.22 In light of

the advantages and disadvantages of MFCs and microalgae, we

expect to establish a symbiotic system, based on the coopera-

tion between microalgae and electrochemically active bacteria,

for simultaneous organic matter, nitrogen and phosphorus

removal and electricity production without energy input.

Although photosynthetic microorganisms have been introduced

to MFCs as oxygen or substrate supply,23,24 organic carbon and

nutrients removal in such a symbiotic system has not yet been

proposed.

By combining microalgae cultivation and MFC technologies,

electrochemically active bacteria in the anode can oxidize organic

matter to release electrons, protons and CO2. Meanwhile, with

solar illumination, microalgae in the cathode will uptake

nitrogen and phosphorus as well as the CO2 released from the

anode for photosynthesis. In return, oxygen produced from algal

photosynthesis can serve as electron acceptor for electricity

generation and possible nitrification process at the cathode.

Through this synergistic interaction, carbon, nitrogen and

phosphorus can be removed from wastewater without extra cost

(e.g., mechanical aeration), while electricity and potentially

valuable algae biomass can be produced simultaneously.

Based on the above hypothesis, in this study, an innovative

sediment-type photomicrobial fuel cell (PFC), which was

a combination of microalgae cultivation and MFC technologies,

was developed for wastewater treatment. Its performance was

investigated in terms of power generation, biomass generation,

C, N and P removal and microbial diversity. This study explores

a better understanding of an algae–electrochemically active

bacteria coupled system and offers new information on cost-

effective wastewater treatment.

This journal is ª The Royal Society of Chemistry 2011

2. Experimental

2.1 Microalgae culture and media

Chlorella vulgaris was purchased from Scandinavian Culture

Collection of Algae & Protozoa (Section for Aquatic Biology,

University of Copenhagen, Denmark) and grown on modified

MWC media at room temperature (22 � 3 �C).25 The algae

suspension was centrifuged (10 000 � g) and washed with DI

water 3 times to remove residual carbon and nutrient sources

before being added to the PFC reactor. The initial concentration

(g dry mass L�1) of Chlorella vulgaris was estimated by optical

density (OD) at 658 nm according to a correlation curve (ESI,

Fig. S1†).

2.2 PFC setup and operation

A sediment MFC was built in a 500 mL glass bottle (Kimax*GL

45 media/storage bottle). The anode, made of carbon paper

(surface area 9 cm2, Toray carbon paper, E-TEK division, USA),

was placed on the bottom. The cathode, a piece of 5% wet

proofed carbon paper containing 0.5 mg Pt cm�2 (surface area 9

cm2, E-TEK), was hung about 5 cm above the anode and

connected to the anode using insulated copper wire (Fig. 1).

Sediment (water content 25%) from Bagsvaerd Lake (55�460N,

12�270E), Denmark, was filled into the glass bottle, producing

a 0.5 cm sediment layer (8% of total reactor volume) above the

Energy Environ. Sci., 2011, 4, 4340–4346 | 4341

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anode. The characteristics of the sediment are shown in Table

S1 (ESI†). The composition of Bagsvaerd Lake water can be

obtained from a previous report.26 Synthetic wastewater con-

taining 197 mg L�1 acetate, 53.1 mg L�1 NH4+–N and 10 mg L�1

PO43�–P was filled in the bottle, reaching a total water volume

of 250 mL. The synthetic wastewater also contained: 1.84 mg

L�1 Na2SeO3$5H2O, 70 mg L�1 MoO3, 40 mg L�1 MnCl2,

44 mg L�1 MgSO4, 15 mg L�1 CaCl2, 2 mg L�1 FeCl2$4H2O, 3.4

mg L�1 MnSO4, and 1.2 mg L�1 (NH4)6Mo7O24$4H2O. The

reactor was started up without algae addition (sediment MFC

mode), and the cathode solution was aerated with air pumps. 10

mL of the synthetic wastewater was added into the reactor once

a day to compensate for the evaporation loss and replenish the

substrate. After start up (approx. 2 months), the reactor was

refilled with the synthetic wastewater. Chlorella vulgaris was

then added to give the desired concentration according to the

following experiments (PFC mode). The PFC reactor was

closed with a rubber stop (no aeration supply), and operated at

batch mode throughout the following tests. Illumination (200

mmol s�1 m�2 on the top of water surface) was achieved via full

spectrum light bulbs, which were controlled by a timer to adjust

the light/dark photoperiod. The dark condition was created by

placing the PFC reactor in a dark room and packed with

aluminium foil. Except for different photoperiod test, the illu-

mination time was 24 hours per day. The cell voltage across

a 1000 ohm resistor was recorded with 10 min interval by

a digital multimeter (Model 2700, Keithley Instruments, Inc.,

Cleveland, OH, USA). For different COD/N experiments, the

acetate concentration was first fixed at about 147.8 mg COD

L�1, while the NH4+–N concentration in the synthetic waste-

water was changed to 30.1, 53.4, and 89.0 mg L�1, resulting in

COD/N of 5.0, 2.9, and 1.7 g g�1, respectively. Besides, acetate

and NH4+–N were also changed to 738.8 mg L�1 and 53.8 mg

L�1, respectively, resulting in a COD/N of 13.7 g g�1. Another

two reactors were set up for control experiments. All

experiments were carried out in duplicate at room temperature

(22 � 3 �C).

2.3 Analytical methods and calculations

Current (I), power (P¼ IV), and Coulombic efficiency (CE) were

calculated as previously described, with the power density

normalized by the projected surface area of the anode.14 Chem-

ical oxygen demand (COD), total suspended solid (TSS),

nitrogen species and P–PO43� were measured according to the

standard method (APHA, 1999). To measure these parameters in

the solutions, the sample was centrifuged (13 000 � g) before

analysis to remove solids; while measuring the total N (or P) in

the biomass, the sample was digested before analysis (APHA,

1999). N2 and CO2 in the headspace were analyzed using a gas

chromatograph (MicroLab, Arhus, Denmark) equipped with

a thermal conductivity detector (TCD). Acetate was measured by

gas chromatography with FID detection (Agilent, 6890) as

previously described.27 pH was measured with a PHM 210 pH

meter (Radiometer). Dissolved oxygen (DO) was measured using

a DO meter (Microprocessor oximeter 539, Germany). OD was

measured by a spectrophotometer (Spectronic 20D+, Thermo

Scientific). Light intensity was measured by a photometer (model

L1-189, L1-COR, USA).

4342 | Energy Environ. Sci., 2011, 4, 4340–4346

2.4 Microbial community analysis

Biomass and water samples were collected from different parts of

the PFC. Total DNA extraction, PCR-DGGE and 16S rDNA

analysis were done as described previously.27 Nucleotide

sequences have been deposited in the GenBank database and are

available under accession numbers JF979184–JF979197.

3. Results and discussion

3.1 Power and algae biomass production

An example of one cycle of power generation and algae biomass

production is shown in Fig. 2a. With an initial algae concen-

tration of 3.5 g L�1, a stable power density of 68 � 5 mW m�2

(0.25 V) was generated and kept for the following 80 h (Fig. 2a).

Along with electricity, algae biomass concentration at the end of

operation increased to 4.06 g L�1. The average biomass

production rate was 0.14 g L�1 d�1. No electricity generation was

observed in the reactor without Chlorella vulgaris addition

(control 1), which might be due to the insufficient electron

acceptor (oxygen) without algae photosynthesis. Similarly, no

appreciable algae biomass generation was observed during open

circuit operation (control 2), which could be due to the insuffi-

ciency of inorganic matter (CO2) for algae photosynthesis, as the

organic matter degradation rate was much slower under open

circuit operation in MFCs.5 The above results indicated that

both algae photosynthesis and exoelectrogenesis were an integral

part of the PFC.

3.2 Organic carbon, nitrogen and phosphorus removal in the

PFC

The carbon, nitrogen and phosphorus removal during electricity

generation was investigated. Only 0.8 mg L�1 of acetate was

detected at the end of operation, resulting in a removal efficiency

of 99.6% (Fig. 2b). Similarly, NH4+–N decreased gradually from

initial 55.6 � 0.1 to 6.6 � 0.1 mg L�1 (Fig. 2c). Meanwhile, 7.8 �0.1 mg L�1 of NO3

�–N and 3.4 � 0.1 mg L�1 of NO2�–N were

detected at 72 h and 48 h, respectively. But they were both

removed afterwards, resulting in 87.6% total nitrogen removal at

the end of batch run (Fig. 2c). The phosphorus removal efficiency

was relatively lower and about 70% of PO43�–P was removed

along with electricity generation (Fig. 2d). Much lower acetate

(max. 38%), NH4+–N (max. 16%) and PO4

3�–P (max. 18%)

removals were observed in the control reactors without algae

addition (control 1) or under open circuit operation (control 2)

(Fig. 2b–d).

A mass balance among COD, nitrogen and phosphorus was

established for elucidating the removal mechanisms. As shown in

Table 1, most of the COD (138.5 mg COD L�1) in the influent

was oxidized and finally captured into biomass at the end of

electricity generation. No CO2 was observed in the headspace,

showing that all the CO2 from substrate degradation was

completely utilized by algae. In addition, methane and hydrogen

were not detected in the headspace, indicating methanogenesis

and hydrogen production had been effectively inhibited in the

system.28

Based on the nitrogen balance, more than 75% of removed

nitrogen (36.4 � 0.1 mg N L�1) was assimilated into algae

This journal is ª The Royal Society of Chemistry 2011

Fig. 2 Performance of the PFC during batch operation: (a) power

density and biomass generation with time; (b) changes of acetate with

time; (c) changes of nitrogen with time; (d) changes of phosphorus with

time. Control 1: no algae addition and control 2: open circuit.

This journal is ª The Royal Society of Chemistry 2011

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biomass. It was also noticed that nearly 22% of removed nitrogen

(10.5 � 0.1 mg L�1) was in the form of N2, which could be due to

nitrification–denitrification taking place in the sediment or

cathode.17,18 Dissolved oxygen in the reactor was lower than 7.0

mg L�1 (Fig. S2, ESI†), at which nitrification and limited elec-

trochemical denitrification in the MFC cathode was observed in

a previous study.19 Identification of nitrifying and denitrifying

bacteria in the reactor (discussed later) confirmed the above

hypothesis. N2O was not measured but based on the nitrogen

mass balance, it was less than 3.2% of total N (1.8 � 0.5 mg L�1).

N2O is a strong greenhouse gas, which accounts for 29.2% of

total nitrogen removal during electrochemical denitrification in

the two-chamber MFC,19 and even as the main product over N2

in the conventional denitrification process.29 The reduction of

N2O in the PFC in this study was due to the direct uptake of most

of the nitrogen by algae biomass, thereby the conventional

nitrogen reduction process (NO3� / NO2

� / N2O / N2)

becoming less dominant. Similarly, more than 93% of removed

PO43�–P (6.9 � 0.1 mg L�1) was assimilated into the algae

biomass (Table 1). The contribution of abiotic phosphorus

precipitation can be negligible due to the relatively low pH (<7.5,

Fig. S2, ESI†) throughout the PFC operation.22 The above

results indicated that the algae biomass uptake was the main

removal mechanism of nitrogen and phosphorus in the PFC.

3.3 Performance of the PFC with different algae

concentrations

The PFC was further tested with different algae concentrations.

The results are presented in Table 2. Power production increased

with the algae concentration and reached to a maximum of 98 �1 mW m�2 at 18 g L�1 algae, which could be due to the increased

oxygen supply by algae. The algae biomass production was not

further improved when the initial algae concentration was higher

than 7.1 g L�1, indicating that other parameters (e.g., illumina-

tion intensity, nutrients concentration) might take over to

determine the algae growth rate.

The removal of carbon and nutrients was variable at different

initial algae concentrations (Table 2). More than 99% of acetate

was removed in all tested algae concentrations. In contrast, only

66.2% of total nitrogen was removed at an algae concentration of

1.8 g L�1, and then it increased to 91.6% at 14.4 g L�1. However,

the removal efficiency was slightly decreased to 86.1%, when 18 g

L�1 algae was applied, which was due to the accumulation of

NO3�–N (5.8 mg L�1) (Table 2). The increased DO might lead

to higher NO3�–N accumulation, while algae utilization of

N–NO3� was a relatively slower process, which requires nitrate

reductase and ATP.30 A significant increase of phosphorus

removal with the algae concentration was observed, indicating

the improved phosphorus uptake at high algae concentration.

3.4 Performance of the PFC at different COD/N ratios

Table 3 summarizes the results of the tests with different COD/N

ratios. No significant differences among power generations were

observed at COD/N ratios ranging from 1.7 to 5.0 g g�1, which

could be due to the same COD concentration (147.8–150.8 mg

L�1) applied. However, a higher power (94 mW m�2) was

observed when a higher COD concentration (738.8 mg L�1) was

Energy Environ. Sci., 2011, 4, 4340–4346 | 4343

Table 1 COD, nitrogen and phosphorus balance

COD Concentration/mg COD L�1 Nitrogen Concentration/mg N L�1 Phosphorus Concentration/mg P L�1

Input Acetate 154.5 � 0.5 NH4+–N 55.6 � 0.1 PO4

3�–P 10.6 � 0.1Acetate 0.6 � 0.1 NH4

+–N 6.6 � 0.1 PO43�–P 3.2 � 0.1

Biomass 138.5 � 1.0 NO3�–N 0.3 � 0.1 Biomass uptake 6.9 � 0.1

Output CH4 —d N2–N 10.5 � 0.1 Othersc 0.5 � 0.1H2 — Biomass uptake 36.4 � 0.1 — —Othersa 15.4 � 1.6 Othersb 1.8 � 0.5 — —

Balance 100% 100% 100%

a Biomass growth in the sediment, calculated according to the balance. b Sediment absorption or N2O. c Sediment absorption. d Below detection.

Table 2 Summary of results obtained under different initial algaeconcentrations, as averages � standard deviations

Algae concentration/g L�1

1.8 7.1 14.4 18.0

CH3COONa mg L�1 Feed 197.0 201.0 199.0 198.0Effluent 1.6 b.d.l. b.d.l. b.d.l.

NH4+–N/mg L�1 Feed 55.6 54.1 53.7 53.8

Effluent 18.8 4.7 2.9 1.7NO3

�–N/mg L�1 Effluentd b.d.l. 0.3 1.6 5.8NO2

�–N/mg L�1 Effluentd b.d.l. b.d.l. b.d.l. b.d.l.PO4

3�–P/mg L�1 Feed 10.6 10.8 10.4 10.5Effluent 5.1 2.7 2.1 1.8

Maximum powerdensity/mW m�2

39.0 76.0 87.0 98.0

Biomassproduction g L�1

0.42 0.67 0.69 0.69

CE (%) 15.4 18.1 17.0 15.6C removala (%) 99.2 100.0 100.0 100.0N removalb (%) 66.2 90.8 91.6 86.1P removalc (%) 51.9 75.0 79.8 82.9

a Evaluated as the ratio of the acetate removed to the acetate fed at thebeginning of the batch. b Evaluated as the ratio of the total N removedto N fed at the beginning of the batch. c Evaluated as the ratio of thetotal P removed to P fed at the beginning of the batch. d N–NO3

� andN–NO2

�1 were not fed in the influent.

Table 3 Summary of results obtained under different COD/N ratios inthe feed, as averages � standard deviations. 7.1 g L�1 algae was applied

COD/N/g g�1

0 1.7 2.8 5.0 13.7

CH3COONa/mg COD L�1

Feed 0 148.5 147.8 150.8 738.8Effluent b.d.l.e b.d.l. b.d.l. b.d.l. 1.2

NH4+–N/mg N L�1 Feed 89.0 89.0 53.4 30.1 53.8

Effluent 87.5 19.7 4.6 2.1 3.5NO3

�–N/mg N L�1 Effluentd b.d.l. 0.5 0.3 0.1 b.d.l.NO2

�–N/mg N L�1 Effluentd b.d.l. 0.1 b.d.l. b.d.l. b.d.l.PO4

3�–P/mg L�1 Feed 10.5 10.4 10.8 10.7 10.0Effluent 10.3 2.1 2.7 3.5 1.9

Maximum powerdensity/mW m�2

3.0 79.0 76.0 78.0 94.0

Biomassproduction/g L�1

0.01 0.72 0.67 0.58 0.72

CE (%) b.d.l. 19.8 18.1 19.6 8.4C removala (%) 0 100 100 100 99.9N removalb (%) 1.7 77.3 90.8 92.7 93.4P removalc (%) 1.9 79.8 75.0 67.2 81.0

a Evaluated as the ratio of the acetate removed in the system to acetate inthe influent. b Evaluated as the ratio of the total N removed to N in theinfluent. c Evaluated as the ratio of the total P removed to P fed at thebeginning of the batch. d N–NO3

� and N–NO2� were not fed in the

influent. e Below the detection limit.

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supplied at a COD/N ratio of 13.7 g g�1, indicating that power

generation was mainly dependent on the substrate concentration

and not the COD/N ratio. No appreciable electricity generation

was observed without substrate supply. The carbon and nutrients

removal efficiency varied according to different COD/N ratios.

Acetate was nearly removed ($99%) at all tested COD/N ratios.

Unlike carbon, the nitrogen removal increased with the COD/N

ratio, and the maximum removal efficiency of 93.4% was ach-

ieved at a COD/N ratio of 13.7 g g�1. However, a decrease of

phosphorus removal efficiency from 79.8 to 67.2% was observed

when the COD/N ratio was increased from 1.7 to 5.0 g g�1 (the N

level was 89.0 to 30.1 mg N L�1, respectively). It was probably

due to the relatively lower nitrogen concentration at high COD/

N ratios, which limited the phosphorus uptake capacity of

algae.31 Interestingly, phosphorus removal (81%) was improved

at a COD/N ratio of 13.7, where much higher COD concentra-

tion was applied compared with other COD/N ratios. More

organic matter supply could lead to more CO2 release through

bacterial oxidation, which might accelerate the algal photosyn-

thesis, and thus, increased nutrients uptake. It was observed that

4344 | Energy Environ. Sci., 2011, 4, 4340–4346

more than 77.3% of nitrogen and 67.2% of phosphorus were

removed even at a relatively low COD/N ratio of 1.7. Normally,

a COD/N ratio over 7 g g�1 is typically required for a conven-

tional (microbiological) nitrogen removal process.2 Therefore,

the PFC has broad applicability for wastewaters containing

various COD/N ratios.

3.5 Effects of light/dark cycles on the PFC performance

The influence of illumination on the PFC performance was

investigated. With 10 h/14 h light/dark photoperiod, power

generation increased when the light was on and decreased in the

dark. However, the maximum stable power density of 61 � 1

mW m�2 was always observed at the end of the light period

(Fig. 3a). The similar phenomenon has also been observed in

photosynthetic organism catalyzed sediment MFCs.24,32 It could

be due to the gradual oxygen accumulation from algal photo-

synthesis during the light period. The biomass concentration

was 4.03 � 0.02 g L�1 at the end of operation, which was close

to the value obtained under continuous illumination shown in

Fig. 2a, but a relative longer time (approx. 140 hours) was

This journal is ª The Royal Society of Chemistry 2011

Fig. 3 Effect of illumination on the performance of the PFC: (a) elec-

tricity generation; (b) biomass production; (c) acetate removal and (d)

nitrogen and phosphorus removal. Initial algae concentration was 3.5 g

Fig. 4 DGGE profile of microbial communities (left) and the schematic

of the sampling sites (right) in the PFC reactor: (A) original inoculum

sediment which was taken before experiments; (B) cathode surface; (C)

middle level water; (D) anode surface. Numbers represented bands that

were excised and sequenced for further analysis.

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required (Fig. 3b). Comparatively, no significant power and

biomass generation was observed at a light/dark period of 0 h/

24 h (Fig. 3a and b).

With 10 h/14 h photoperiod, more than 99% of acetate was

degraded (Fig. 3c). Meanwhile, NH4+–N was decreased from

54.1 to 5.9 mg L�1, resulting in a total nitrogen removal effi-

ciency of 89% (Fig. 3d). Similarly, PO43�–P was gradually

decreased from 10.2 to 3.8 mg L�1 (Fig. 3d). The only differ-

ence here, compared with continuous illumination, was the

longer reaction time. No appreciable carbon, nitrogen and

phosphorus removal was observed during the photoperiod of

0 h/24 h (total dark) (Fig. 3c and d), indicating the importance

of illumination. Furthermore, the above results showed the

applicability of the PFC in regions with year-round solar

radiation, as the 10 h/14 h photoperiod is close to the natural

sunlight condition.

3.6 Microbial community

Bacterial community profiles of the PFC were analyzed by

DGGE (Fig. 4), and phylogenetic affiliations of the representa-

tive band sequences were shown as 16S rDNA genes library

(Table S2, ESI†). The bacterial communities in the PFC showed

to be notably different from the original inoculated sediment.

Furthermore, the samples taken from different sites (anode in

sediment, liquid and cathode) had different community compo-

sitions. On the cathode surface, bacteria belonging to uncultured

L�1. Day/night mode: 10 h/14 h photoperiod; dark (all night) mode: 0 h/

24 h photoperiod. L/D: light/dark mode; D: dark mode.

Energy Environ. Sci., 2011, 4, 4340–4346 | 4345

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Flavobacteria and Sphingobacteria (Fig. 4, bands 9 and 10,

respectively) were detected as dominant species. The former one

was isolated from a denitrifying bioreactor, while the latter was

taking part in ammonia oxidation. Similarly, uncultured Fla-

vobacteria were also detected in the middle-level water, which

was otherwise, dominated by the bacteria similar to Terrimonas

ferruginea sp. and Bradyrhizobium sp. In contrast, the bacterial

community from the anode was dominated by iron reducing

(band 13) and denitrifying bacteria (band 14), which belong to

Alphaproteobacteria. Identification of ammonia oxidizing and

denitrifying bacteria in the reactor verified thereby the previous

hypothesis that the nitrogen gas was generated by a nitrification–

denitrification process.

The purity of algal species in the system was not analyzed, but

it is reasonable to assume that Chlorella vulgaris was at least the

dominant species because the artificial wastewater and algae were

refilled at each batch.

4. Conclusion

In summary, the PFC system described in this paper was able to

produce electricity and algal biomass, and remove/capture

carbon, nitrogen and phosphorus simultaneously. To the best of

our knowledge, this work represents the first comprehensive

study of wastewater nutrients removal along with energy gener-

ation through the symbiotic interaction between electrochemi-

cally active bacteria and microalgae. Employment of the closed

system in this study was able to easily track the fate of C, N, and

P, which was helpful for better understanding of the wastewater

removal mechanism. This work serves as a state-of-the-art study

about integration of MFCs with algae technology for wastewater

treatment. Further implementation of this concept with an open

system may promote its practical application. Future studies for

development of economical and efficient algae harvesting tech-

nologies are necessary in order to further use the produced algae

biomass for bioenergy production.

Acknowledgements

The authors thank Hector Garcia for his help with analytical

measurements. The authors also thank Susan Løvstad Holdt and

Poul Møller Pedersen for algae ordering and advice. This study

was funded by the Ministry of Science Technology and Inno-

vation, the Strategic Research Program for Health, Food, and

Welfare, Ref. no. 09-067601.

4346 | Energy Environ. Sci., 2011, 4, 4340–4346

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