Research Article Bioelectricity Generation by Microbial ...bioaugmentation voltage maxima...

©Journal of Applied Sciences & Environmental Sustainability 3 (6) 10 - 22, 2017 e-ISSN 2360-8013

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Research Article

Bioelectricity Generation by Microbial Augmentation of Animal Wastes

1,2*Ajunwa, O.M. 1Odeniyi, O.A., 3,4Audu, J.O. and 1Onilude, A.A.

1Department of Microbiology, University of Ibadan, Ibadan, Nigeria 2Department of Microbiology, Modibbo Adama University of Technology, Yola, Nigeria 3Department of Laboratory Technology, Modibbo Adama University of Technology, Yola, Nigeria 4Faculty of Bioscience and Medical Engineering, Universiti Teknologi Malaysia, Malaysia

Corresponding Author: [email protected]

ARTICLE INFO

Article history

Received: 01/01/2017

Accepted: 15/03/2017

A b s t r a c t

Bioelectricity generation by microbial activity on substrates using a bioelectrochemical

system is a new form of bioenergy being developed based on green energy demands.

Animal wastes serve as a veritable source of cheap feedstocks for bioelectricity generation.

Bioaugmentation of inherent microbial communities by introduction of external efficient

species has been a well-known system of upgrading bioactivity within substrates.

Microbial loads of cow dung and poultry manure were assessed by sampling total viable

counts on the 1st, 10th, 20th and 30th days. A closed circuit voltage system with the

connection of a 100Ω resistor to a bioelectrochemical system was used for bioelectricity

measurements. Cow dung and poultry manures were applied in bioelectricity generation

for a 30day period at room temperature. The wastes were further augmented with

electrogenic isolates Pseudomonas sp MT08 and Enterobacter sp MT031 in single and co-

culture, and their bioelectricity generation was monitored in comparison with non-

augmented substrates. Non-augmented set ups using unsterilized cow dung and poultry

manures yielded maximum voltages of 315.20±3.1mV and 209.12±2.13 mV, while the set-

ups with sterilized animal wastes yielded no voltages, and subsequently no current, thus

showing the microbial origin of bioelectricity in the set-ups. In single culture

augmentations the highest voltage maxima of 682.32 ± 1.42mV was observed from the

set-up of cow dung augmented with Pseudomonas sp. Augmentation using the

Pseudomonas and Enterobacter species co-culture, resulted in an increased voltage

maxima value of 1585.31±1.25 mV when used on cow dung slurry. The lowest

bioaugmentation voltage maxima measurement of 301.10±0.96 mV was observed with the

single application of Enterobacter sp and poultry manure. Bioelectricity from microbial

augmented animal wastes and other wastes can be applied in small scale energy generation

systems.

© Journal of Applied Sciences & Environmental Sustainability. All rights reserved.

Bio-augmentation; Animal wastes; Bioenergy; Electrogenic bacteria

mailto:[email protected]


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1. Introduction

Global energy demands are on a geometric increase, with a very obvious dependence on fossil fuels which

can result to the next level of depletion of already limited reserves as well as leading to negative effects of

carbon emissions on the environment (Amann, 1996; Das and Veziroglu, 2001; Mohan et al., 2008). The

consequent effects of this lopsided energy dependence have also led to issues of climate change with

regards to global insecurity and safety, thus a holistic search for alternative and complementary energy

sources (Logan, 2008). Attention is being given at present to green and sustainable sources of energy that

can be renewable with less deleterious effects on the environment. Bioenergy from organic matter has been

a major form of this green energy researched upon over the years; a new form of which is Bioelectricity -

which involves the direct production of electricity from microbial biomass (Logan, 2008; Kracke, et al.,

2015).

Diverse forms of substrates have been used in bioelectricity generation. Logan et al (2005), used cysteine as

a substrate for bioelectricity generation after obtaining the microbial isolates from marine sediment. Starch

was also used in electricity generation, and electrogenic microorganisms from waste water were applied

(Methe et al., 2003; Kim et al., 2004). A combination of glucose and glutamic acid was tried as substrates

by Phung et al (2004), and the ability to optimize the activities of groups of alpha-, beta-,

gammaproteobacteria and bacteriodetes in bioelectricity generation was determined. River water has also

been employed as a substrate as reported by Phung et al. (2004) and electrogenic bacteria were tested for

bioelectricity generation. In most cases, direct use of an extraneous material containing a complex

community of microorganisms as inoculum (without isolation of the electrogenic species) was adopted.

River sediments, marine sediments, and waste water have all been used to inoculate bioelectrochemical

systems for bioelectricity generation (Lee et al., 2003; Methe et al., 2003; Kim et al., 2004; Phung et al.,

2004; Logan, 2005) without isolating the electrogenic species. This limits the knowledge of specie based

electrogenicity and causes fallacious inference in the monitoring of electrogenic species and their

mechanisms of action. Optimisation of electricity generation from microbial species is most possible when


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the species have been identified and their basic electrophysiology understood. This assists in metabolic

manipulations of the isolates as well as maximization of their electrogenic potentials (Logan, 2008).

Bioaugmentation involves the use of selected efficient microbial strains to improve the processes inherent

within a given natural microbial system harbouring a mixture of both indigenous microbial flora and natural

substrates (Bitton, 2005). Bioaugmentation of autochthonous microbial flora within a natural system has not

been deeply exploited with respect to microbial electricity generation, as there are very few researches to

substantiate this view (Logan, 2008). There are positive potentials for increased electricity yield when

microbial augmented bioelectrochemical systems are used in comparison with non-augmented systems or

single species of bacteria. This is due to the factor of higher nutrients, higher microbial load, and a better

interspecies collaboration leading to higher electrogenicity (Logan and Regan, 2006).

Animal wastes possess high microbial flora as well as contain nutrients for their metabolic activities. They

are a good example of substrates carrying autochthonous microbial species as well as a nutrient table for the

microorganisms to thrive. Cow dung and poultry manure are one of the two readily available animal wastes

in the country and a system of bioresource management which involves their reuse for energy purposes is

advocated. They can be a veritable source of both nutrients and substrates that can be bio-augmented to

yield increased electricity generation (Ofoefule et al., 2010; Godi et al., 2013; Asikong et al., 2014;

Okoroigwe et al., 2014).

This work thus aims to assess the bioelectricity production potentials of cow dung and poultry manure as

animal wastes augmented with efficient electrogenic microorganisms in comparison with the non-

augmented experimental set ups.


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2. Materials and methods

2.1 Sample collection and preparation

Fresh cow dung and poultry droppings used in this study were obtained from the school farm of a

university. The samples were collected in clean polyethylene bags and transported to the laboratory for

further conditioning and analysis. The animal wastes were separately made into a slurry with equal volumes

of distilled water in the ratio 1: 2 according to the method of Rabah et al. (2010), and subsequently used in

the bioelectrochemical reactors.

2.2 Microbial load determination

About 200ml each of the cow dung and poultry manure slurries were transferred into 250ml conical flasks

and incubated at room temperature (28±2oC). Samples were subsequently taken at different times (1st day,

10th day, 20th day and 30th day) for determination of microbial load. The samples were serially diluted (10

fold) and plated out onto nutrient agar plates. The plates were incubated for 48h and colonies were

subsequently counted.

2.3 Microorganisms

Electrogenic isolates Pseudomonas sp MT 08 and Enterobacter sp MT 031 identified from earlier

experiments (yet to be published data) were obtained and used for the augmentation studies. They were

characterized as high anodic biofilm producers and efficient electrogenic strains. The isolates were

suspended in nutrient broth at 4oC until used. Inoculums were prepared by standardization of the cells to 102

CFU/ml.

2.4 Bioelectrochemical reactor set-up

The bioelectrochemical reactor used in this study was a batch culture operated 2-chambered polyethylene

teraphtalate reactor with an adjoining proton exchange membrane (Figure 1). It had graphite electrodes

(anode and cathode), with 500ml anodic and cathodic chamber volume, 74.22.cm2 electrode surface area,


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copper wire extensions connecting the electrodes to a digital multimeter (model DM-87, HTC

Instruments®), 50cm3 agar-agar membrane. The polyethelene teraphtalate reactor was sterilized according

to the protocol of Sharma (Sharma et al., 2012) by a combining treatment with 75% ethanol and ultraviolet

radiation (254nm UV-C; dose – 5000µW.s/cm2) for 30 minutes at an exposure distance of 10cm. The cow

dung and poultry manure slurry served as the anolyte with augmentation by standardized electrogenic

isolates (102 CFU/ml). A preparation of 0.5% potassium permanganate solution was used as the catholyte

2.5 Bioelectricity measurements

Bioelectricity production was measured by taking closed circuit voltage readings using the digital

multimeter, and a 100Ω resistor. To determine the viability of the bioprocess, current was calculated using

Ohm’s law (the formula Voltage = Current x Resistance). Voltage plots against reaction time (hours) were

measured and used as the volume of bioelectricity generated (Mohan et al., 2008).

Figure 1: Bioelectrochemical reactors used in bioelectricity generation by microbial augmentation of cow dung

and poultry manure.


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3. Results and Discussion

The total viable count from animal waste samples (Table 1) shows that the cow dung had higher counts (9.4

x 1010 CFU/g) when compared with the value for poultry manure (6.4 x 109 CFU/g). Both substrates

showed highest values of bacterial counts on the 20th day of sampling. Figure 2 shows the bioelectricity

yield in milli volts (mV) of the augmented and non-augmented set-ups. Comparative assessments of the

bioelectricity production potentials of sterilized animal wastes, unsterilized animal wastes, and unsterilized

animal wastes augmented with Pseudomonas sp MT 08 and Enterobacter sp MT 031 respectively showed

that the augmentation of unsterilized cow dung with Pseudomonas sp had the highest maximum voltage of

682.32 ±1.42mV. The set up of unsterilized poultry manure augmented with Pseudomonas sp yielded a

maximum voltage value of 590.1±2mV, which was followed by 400.02±1.1mV and 301.0±0.82mV yielded

by Enterobacter sp augmentations of unsterilized cow dung and unsterilized poultry manure respectively.

Maximum voltage yields for non-augmented set ups were however lower in comparison with augmented set

ups as the values of 235 ±0.32 mV and 203.05±0.1mV were obtained as maximum voltage yields for

unsterilized cow dung and poultry manures respectively. Zero voltage values were obtained with set ups of

sterilized cow dung and poultry manure.

There was a variation in the time the maximum voltages were achieved for all the set ups. For the setup of

unsterilized cow dung augmented with Pseudomonas sp, the maximum voltage was achieved on the 29th

day, while the maximum voltage for the setup of unsterilized poultry manure augmented with Pseudomonas

sp was recorded on the 30th day. When Enterobacter sp was used to augment the animal wastes, the

maximum voltage was obtained on the 28th day for the two wastes. The non-augmented cow dung showed

maximum voltage on the 30th day while maximum voltage was obtained by the non-augmented poultry

manure on the 29th day.

The result of the set up carrying animal wastes augmented with the co-culture of Pseudomonas sp and

Enterobacter sp showed that the maximum voltage of 1586.67±0.45mV and 1111.1±0.32mV for


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augmentations with cow dung and poultry manure respectively. The maximum voltage values were

recorded on the 27th day for cow dung and on the 29th day for poultry manure.

Table 1: Total viable count from cow dung and poultry manure

Sampling time

(Days)

Total Viable

Count

in Cow dung (CFU/g)

Total Viable

Count

in Poultry manure (CFU/g)

1 3.5 x 106 2.2 x 105

10 5.1 x 108 4.0 x 107

20 9.4 x 1010 6.4 x 109

30 4.1 x 104 3.0 x 103

Key: CD1 – unsterilized cowdung, CD2 – sterilized cowdung, PM1 – unsterilized poultry manure, PM2 – sterilized

poultry manure.

Figure 2. Comparative bioelectricity yield (mV) of sterilized animal wastes, unsterilized animal wastes, and

unsterilized animal wastes augmented with Pseudomonas sp MT 08 and Enterobacter sp MT 031

respectively.


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Key: CD 1 – unsterilized cow dung, PM 1 – unsterilized poultry manure

Figure 3. Bioelectricity yield (mV) of unsterilized animal wastes augmented with co-culture of Pseudomonas sp

MT 08 and Enterobacter sp MT 031

The results showed there was a higher microbial load of cow dung slurries compared with the poultry

manure. The presence and growth of the microorganisms found within the substrate showed that the

substrate type had an effect on the microbial flora. Asikong et al. (2014), stated that there was a greater

value of nutritive components present within cow dung in comparison with poultry manure. This could lead

to a greater diversity of autochthonous microbial flora in the cow dung compared with the poultry manure.

There was consequently a higher yield in bioelectricity when the cow dung was used as substrate in

comparison with the poultry manure substrates. This verifies the relevance of load of indigenous

microorganisms thriving as a result of the higher nutritive components inherent in cow dung when

compared with poultry manure, and the directly proportional relationship with increased bioelectricity yield.

Logan (2008) stated that there was a directly proportional correlation between bioelectricity yields and

microbial load within substrates. The reduction in microbial load towards the 30th day of sampling the

animal wastes led to a little reduction in bioelectricity generated in the set ups between the 27th and 30th day

of bioelectricity measurements.


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There were bioelectricity yields with set ups containing unsterilized animal wastes, but zero bioelectricity

yield with set ups containing sterilized cow dung and poultry manure. This confirms the microbial origin of

the bioelectricity as the metabolic activities of the microorganisms inherent within the animal wastes were

responsible for electrogenicity and bioelectricity generation. Rabah et al. (2010) and Ofoefule et al. (2010),

established the facts that animal wastes harboured a large presence of energy-relevant microbial species that

are naturally present within the waste samples, thus an unsterilized/raw sample of animal wastes has

potentials of yielding a quantity of bioenergy depending on the metabolic state of existing microorganisms

within the animal waste.

The augmentation of the animal wastes with earlier determined electrogenic microbial species led to an

increase in bioelectricity generation in comparison with the non-augmented set ups thus validating the

relevance of bioaugmentation as a means for increasing bioactivity within a specific system. The set ups of

animal wastes augmented with Pseudomonas sp gave a higher yield in comparison with the set ups

augmented with Enterobacter sp. This showed that the Pseudomonas sp augmented systems were more

efficient most probably because of the higher electrogenic activity of Pseudomonas sp MT08 in comparison

with Enterobacter sp MT031 (yet to be published research work). Bioelectricity values independently

obtained by Rabaey et al. (2005) and Feng et al. (2014) also showed that there was higher yield of

Pseudomonas sp set ups when compared with the set ups of Enterobacter species. They attributed this to

their different metabolic functionalities with respect to electrogenicity, and their unique electron

transport/export systems.

Bioaugmentation using a co-culture of electrogenic Pseudomonas sp MT08 and Enterobacter sp MT031

was more efficient that single culture bioaugmentation. This showed that there was an obvious synergy

between the individual organisms that positively affected the bioelectricity yield. The positive electrogenic

potentials of the two electrogenic organisms were highly complementary owing to the fact that inter species

cooperation targeted at higher electrogenicity and bioelectricity yields were responsible. Logan (2008)


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affirmed this observation as it was determined that a better interspecies collaboration between proven

electrogens existed in mixed cultures thus leading to higher bioelectricity generation.

4. Conclusion

The potentials for bioelectricity are still being explored and developed. The ability to utilize wastes that

abound in our environment is a major factor that can step up its application and commercialization process

because of the ease of obtaining such substrates at relatively low costs. Animal wastes are a good example

of environmental wastes that can be bioconverted to energy. Researches on bioenergy from animal wastes

(Ofoefule et al., 2010; Godi et al., 2013) stated that animal wastes are viable environments for microbial

activity and can be readily harnessed for bioenergy directly or indirectly. The use of cow dung and poultry

manure in bioelectricity generation in this study affirmed the point. The utilization of animal wastes stands

as a major form of bioresource management (Asikong et al., 2014; Okoroigwe et al., 2014).

Logan and Regan (2006) also stated that the factors of higher nutrients, higher microbial load, and a better

interspecies collaboration led to higher electrogenicity. The augmentative blends of efficient electrogenic

strains co-cultures, high nutritional composition of substrates and functionally conducive environments for

indigenous microbial flora to thrive can lead to enhanced bioelectricity generation bringing the technology

closer to more effective applications.

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