anaerobic digestion and in situ electrohydrolysis of dairy bio-sludge

Biotechnology and Bioprocess Engineering 15: 520-526 (2010)

DOI 10.1007/s12257-009-0220-y

Anaerobic Digestion and In situ Electrohydrolysis of Dairy Bio-sludge

Krishnan Vijayaraghavan and G. K. Sagar

Received: 18 August 2009 / Revised: 18 November 2009 / Accepted: 5 December 2009

© The Korean Society for Biotechnology and Bioengineering and Springer 2010

Abstract A novel treatment method based on anaerobic

digestion and in-situ electrohydrolysis of dairy bio-sludge

was investigated in this article. The electrohydrolysis was

carried out inside the anaerobic reactor using graphite

anode and stainless steel cathode. The electrons released by

the graphite anode combines with the proton released due

to electrohydrolysis of fatty acids which resulted in the

formation of hydrogen gas. The experiments were con-

ducted using a DC power source under continuous and

intermittent mode of input voltage ranging from 0.5 to

2.5 V for varying influent volatile solids concentration at a

pH 5.3 ± 0.2. The results favored intermittent mode of

input voltage rather than continuous supply. For an influent

total solid concentration of 7% (64,120 mg/L VS), inter-

mittent input voltage of 2 V, and a hydraulic retention time

of 15 days resulted in a volatile solids and soluble COD

removal efficiency of 83 and 74%, while the cumulative

gas generation was 1,051 L with a hydrogen content of

72%.

Keywords: anaerobic digestion, electrohydrolysis, hydro-

gen, bio-sludge, dairy, biomass

1. Introduction

Due to energy crisis and population raise, the world had

started to experience the potentially crippling energy crisis

in power, manufacturing, transportation, heat, and other

needs. Burning of fossil fuels has resulted in the release

of carbon dioxide and combustion pollutants which has

caused enough damage to the global climate. The means of

preventing the catastrophes like energy scarcity and

environmental ruin are still not clear, but one part of the

solution may sure lie in microbial technology. Microbial

energy conversion technology makes use of microorganisms

in churning out organic matter into fuels or to harvest elect-

rons from biomass to generate an electric current, thereby

transforming chemical energy into electrical energy. This

futuristic bio-hydrogen energy technology shall become

reality in the near future [1].

Bioelectrochemical treatment technologies generally

follow either of the following types (i) microbial fuel cells

(MFCs) for electricity production [2] and (ii) biocatalyzed

electrolysis or bio-electrochemically assisted microbial

reactor (BEAMR process) for hydrogen production. Dark

fermentation of glucose produced 4 moles hydrogen and 2

moles of acetate per mole of glucose, whereas biocatalyzed

microbial electrolysis produced 12 moles hydrogen per

mole glucose on theoretical basis [3]. In the case of bio-

catalyzed electrolysis, hydrogen is produced at the cathode

with platinum as a catalyst. External power sources are

required to realize the produced hydrogen. Biocatalyzed

electrolysis of acetate at an applied voltage of 0.5 V pro-

duced 0.02 m3 H2/m3.day with an overall efficiency of

53 ± 3.5%. Under optimized condition a volumetric hydro-

gen production rate of about 10 m3 H2/m3.day was achiev-

ed with 90% efficiency at an applied voltage of 0.3 ~ 0.4 V

[4]. Acetate oxidation in microbial fuel cell also uses the

same concept except that electrical energy was harvested

through an external resistance [5].

Carbohydrates conversion into electricity in a MFC

showed removal efficiency up to 50% of the substrate

COD as current, with a concomitant power generation of

49 W/m3 based on net anodic compartment. As 52% of the

anode compartment was occupied with graphite granules,

the former value corresponded to a power generation of

32.5 W/m3 [6]. Microbial biocathodes hold great promise

as an alternative to platinum, as they can apply inexpensive

Krishnan Vijayaraghavan* and G. K. SagarDepartment of Biotechnology, Biotechnology Research Division, PrathyushaInstitute of Technology & Management, Tamil Nadu 602025, IndiaTel: +91-44-27620450; Fax: +91-44-27620331E-mail: [email protected]

RESEARCH PAPER

Anaerobic Digestion and In situ Electrohydrolysis of Dairy Bio-sludge 521

electrode material like graphite and due to self-regene-

rating capacity [7]. Microbial biocathode concepts have

already been tested successfully for redox cycling of transi-

tion metals (e.g., Mn and Fe) between the cathode and

metal-oxidizing bacteria [8,9], direct electron transfer by

electrochemically active micro-organisms [10] and cathodic

hydrogen production by enzymatic method [11-13]. The

cathodic hydrogen production was investigated using

microbial biocathode consisting of immobilized culture of

Desulfovibrio vulgaris and methyl viologen as redox

mediator [14,15].

In this article the advantage of anaerobic digestion coup-

led with electrohydrolysis were exploited in hydrolyzing

the volatile solids present in the dairy bio-sludge into gas

end product namely hydrogen. The objective of this study

is to investigate the effect of operating parameters such as

influent volatile solids and hydraulic retention time on

hydrogen gas production during anaerobic digestion and

subsequent electrohydrolysis of accumulated fatty acids

based on the mode and value of the input DC voltage.

2. Materials and Methods

2.1. Anaerobic digester

The experimental set-up of the anaerobic digester are as

shown in Fig. 1 had a working liquid volume of 40 L. The

reactor was operated on up-flow mode and is of complete

mixed type. The reactor unit consists of PVC column with

the following dimension 300 mm ID × 700 mm height. A

graphite rod of 290 mm in length and 50 mm in diameter

was used as an anode. Perforated stainless steel sheets

290 mm long, 50 mm wide, and having a thickness of

0.8 mm were used as cathodes. The graphite anode was

surrounded by two cathode sheets. The distance between

anode and cathode was 40 mm. The external power input

in the electrohydrolysis process was achieved using a DC

power source in the range between 0.5 and 2.5 V. During

the anaerobic digestion process the volatile solids present

in the dairy bio-sludge are converted into intermediates like

fatty acids, which are in turn used by the hydrogen gene-

rating bacteria to produce gaseous hydrogen as the end

product. The accumulated fatty acid concentration under-

goes electrohydrolysis in the anode region. The proton

released from the electrohydrolysis of fatty acids (2HAc →

2Ac− + 2H+) combines with the electron yielding hydrogen

gas at the cathode (2H+ → 2e− + H2).

2.2. Analytical process

The soluble COD concentration of the dairy effluent treat-

ment plant bio-sludge sample was determined by chemical

oxygen demand (COD) method by using the filtered

sample with a membrane filter having a pore size of 2 µm.

The chemical oxygen demand measures the amount of

oxidant (Cr2O7

2−) that reacts with the sample under cont-

rolled condition, while the quantity of oxidant consumed

was measured by colorimetric method and expressed in

terms of its oxygen equivalence [16]. The total Kjeldahl

nitrogen was determined by converting amino nitrogen of

organic material and free ammonia to ammonium under

acidic condition. After the addition of the base, the

ammonia was distilled from the alkaline medium and

absorbed in boric acid and determined by titration with

mineral acid. The volatile fatty acid content was deter-

mined by distillation method. The distillation technique

recovers acids containing up to six carbon atoms and the

results are reported on the basis of acetic acid. The total

solids were determined by evaporating the well mixed

sample in a weighted crucible and dried to constant weight

in an oven at 105°C. The increase in weight over that of the

empty crucible represents the total solids. The volatile

solids were determined by igniting the residue that was left

out after the determination of total solids at 550°C. The

weight loss after the ignition represents the volatile solids

content. The oxidation and reduction potential of the

anaerobic digester was measured using redox meter [17].

The hydrogen and methane content in the biogas were

determined by Dräger Method [18].

1. Feed tank 6. Anaerobic digester2. Feed and recycle pump 7. DC power source3. Graphite (Anode) 8. Gas outlet4. Stainless steel (Cathode) 9. Gas meter5. Outlet 10. Recycle line

Fig. 1. Experimental set-up of electro-assisted microbial bio-hydrogen generation.

522 Biotechnology and Bioprocess Engineering 15: 520-526 (2010)

2.3. Preservation of bio-sludge

The raw bio-sludge was collected from the sludge drying

bed of Aavin Dairy Industry of Madhavaram, Chennai

which had the following characteristics as shown in Table

1. The bio-sludge was preserved at a temperature less than

4oC but above freezing in order to prevent the putrefaction

due to microbial action [17].

2.4. Hydrogen generating microflora

The anaerobic reactor was seeded with the hydrogen gene-

rating microflora, which was isolated from the anaerobic

digester of dairy effluent treatment plant based on pH

adjustment at 5 ± 0.1 as stated elsewhere by Vijayaraghavan

et al. [19]. The isolated microflora showed a hydrogen

generating ability of 58 ± 3% during the isolation process

when the experiments were conducted in 250 mL vials at

pH 5.3. The isolated hydrogen generating microflora was

initially grown in 3 L fermenter using dairy effluent as

substrate for a period of 30 days. Thereafter the biomass

was transferred into the anaerobic reactor at a mixed liquor

volatile suspended solid (MLVSS) concentration of 4,800

mg/L.

2.5. Seeding and acclimatization

The start-up operation was carried out by seeding 3 L of

hydrogen generating micro flora with an mixed liquor

volatile suspended solid concentration of 4,800 mg/L into

the anaerobic reactor, to which 10 L of 1% dairy effluent

treatment plant bio-sludge was added and incubated for a

period of 10 days. Thereafter at an interval of 10 days, 10 L

of 1% bio-sludge was added for three consecutive period

in-order to achieve a 100% digester volume operating

efficiency, which marked the end of acclimatization

operation. During the acclimatization period the pH, redox

potential, biogas generation, and its hydrogen content were

monitored.

2.6. Effect of applied voltage on electrohydrolysis

The effect of applied voltage on biogas production and its

hydrogen content was investigated under varying applied

voltage namely 0.5, 1.0, 1.5, 2, and 2.5 V respectively

under two mode of operation viz: (a) continuous supply of

voltage and (b) intermittent supply of voltage at an alter-

nating 1 h of on and off period. Based on the optimized

mode of voltage supply and its quantity, regular treatment

of bio-sludge was investigated using an influent total solids

content of 5 and 7%. The efficiency of the anaerobic

digestion and in-situ electrohydrolysis treatment process

was monitored based on the amount of volatile solids

destroyed, soluble COD removal percent, digester VFA

content, biogas generation, and its hydrogen content.

3. Results and Discussion

3.1. Seeding and acclimatization

Fig. 2 shows the biogas generation and its hydrogen

content during the acclimatizing period using 1% total

solid (TS) of dairy bio-sludge. As the acclimatizing period

increased the biogas generation showed a gradual rise. For

example at the end 20, 40, and 50 days of operation the

biogas generation was found to be 9.8, 11.6, and 12 L/day

with a hydrogen content of 62, 64, and 64% respectively.

When the operating period was increased above 40 days

the gas production showed a stabilized value which con-

firmed the stabilization of anaerobic digestion process and

even the hydrogen content was stabilized at 64%. Hence

at the end of 50th day the acclimatization process was

completed and experiments were conducted in order to

determine the effectiveness of the electro-assisted anaerobic

digestion process. During the acclimatization period

the reactor pH was found to be 5.3 ± 0.2, redox potential

−354 ± 12, and volatile fatty acids (VFA) 1230 ± 34 mg/

L. The reason for choosing acidic pH was to prevent the

growth of methanogenic bacteria in the digester, as hydro-

gen serves as substrate for the methanogenic bacteria

which was in agreement with Lay [20], Oh et al. [21], and

Cohen et al. [22].

Table 1. Characteristics of raw dairy bio-solids

Parameters* Concentration

pH 7.8 ± 0.2

Total solid 9.2 ± 3%

Volatile solid 89 ± 2%

Soluble COD 37,254 ± 187 mg/L

TKN 0.075 ± 0.004%

*pH is a dimensionless parameter.

Fig. 2. Average biogas generation and its hydrogen content duringacclimatizing period (influent TS concentration of 1%).


3.2. Optimizing electrohydrolysis voltage

The effect of electrohydrolysis input voltage on biogas

generation and its hydrogen content was investigated at

0.5, 1.0, 1.5, 2, and 2.5 V at an influent total solid (TS)

concentration of 2%, pH 5.1 ± 0.2 at 10 day hydraulic

retention time (HRT) for a period of 30 days. As shown in

Fig. 3 with the increase in input voltage the biogas

generation also showed an improved yield. For example at

an input voltage of 0.5, 1.0, 1.5, 2, and 2.5 V when the

reactor was operated for a period of 30 days the biogas

generation was found to be 10.9, 13.5, 17.0, 22.2, and

23.8 L/day, respectively. Irrespective of the input voltage

the hydrogen content was found to be 68 ± 2%. While in

the absence of external voltage the biogas generation was

found to be 9.7 L/day with a hydrogen content of 61%. In

general with increase in input voltage the biogas generation

value also showed an increase, while at an input voltage of

2 and 2.5 V a marginal raise in biogas value was observed.

Hence an applied voltage of 2 V was chosen as an

optimum voltage in this present investigation.

3.3. Effect of continuous and intermittent electrohydro-

lysis

Based on the optimized electrohydrolysis voltage, the

effect of continuous and intermittent power supply was

investigated (Fig. 4). The experiments were carried out at

an input voltage of 2 and 2.5 V for an influent total solid

content of 2%. The results showed that instead of continu-

ous supply of electrohydrolysis voltage to the anaerobic

digestion, an alternating power supply for every 1 h to the

reactor showed an improvement in the volatile solids

destruction efficiency. For example in the of case anaerobic

digestion at 10 day hydraulic retention time when followed

by in situ electrohydrolysis at a continuous input voltage of

2 and 2.5 V resulted in a volatile solids destruction of 77

and 83%. Whereas for the above said hydraulic retention

time and input voltage in the case of intermittent power

supply for an alternating 1 h period resulted in a volatile

solids destruction efficiency of 82 and 86% respectively.

At an intermittent applied voltage the removal efficiency of

VS was marginally higher when compared with the

continuous supply of applied voltage. Table 2 shows the

mode of reactor operation and its VFA content at 2% TS of

dairy bio-sludge. As the VFA content in the reactor was

low in the case of continuous electrohydrolysis it can be

confirmed that the biological reactions are hindered.

Figs. 5 and 6 shows the corresponding biogas generation

and its hydrogen content during continuous and inter-

mittent power input to the anaerobic reactor at 2 and 2.5 V.

Similar to volatile solids destruction efficiency the biogas

generation also showed a marginal higher value during

intermittent power supply at higher input voltage. The

results showed that for an influent TS content of 2% at

continuous and intermittent power supply of 2 V and a

hydraulic retention time of 10 days resulted in a biogas

production of 19.5 and 23 L/day with a hydrogen content

of 67 and 70%. Whereas for the above said condition with

2.5 V of continuous and intermittent power supply resulted

in biogas generation of 20.2 and 24.6 L/day with an

average hydrogen content of 69 and 71% respectively.

Fig. 3. Biogas generation versus operating period during varyinginput voltage (influent TS concentration of 2%).

Fig. 4.Volatile solids destruction efficiency versus operating periodunder continuous and intermittent power input of 2 and 2.5 V(influent TS concentration of 2%).

Table 2. Mode of reactor operation and its VFA content at 2% TSof dairy bio-sludge

Operating type VFA content (mg/L)

Anaerobic digestion 1,980

Anaerobic digestion coupled with continuous in-suite electrohydrolysis

90

Anaerobic digestion coupled with in-suite electrohydrolysis

310


3.4. Anaerobic digestion coupled with intermittent

electrohydrolysis

The treatment efficiency of anaerobic digestion coupled

with intermittent electrohydrolysis on dairy bio-sludge was

investigated for an influent total solid content of 5 and 7%.

At an influent TS concentration of 5% and a hydraulic

retention time of 10 and 15 days resulted in a VS removal

efficiency of 72 and 92%. In the case of 7% influent TS

concentration for the above said hydraulic retention time

resulted in a VS destruction efficiency of 69 and 83%

respectively. As corroborating evidence the biogas gene-

ration (Fig. 7) also showed an increased yield at higher

influent VS concentration and hydraulic retention time. At

an influent TS concentration of 5% and a hydraulic

retention time of 7 and 10 days the cumulative biogas

generation was found to be 559 and 978 L, while at 7%

influent TS concentration for the above said condition the

cumulative biogas generation was found to be 612 and

1,051 L respectively. The hydrogen content in the biogas

had a value ranging between 62 and 72% which confirmed

the stability of microflora during the degradation of dairy

bio-sludge. Hence it can be concluded that the metabolic

reaction of the hydrogen generating bacterial species

occurred in a steady phase with that of electrohydrolysis

process. Similar observations were made by Chang and

Lin [23] who stated that longer hydraulic retention time

favored degradation but did not alter the hydrogen pro-

duction. Whereas Van Ginkel et al., 2005 [24] stated that

nutrient addition resulted in more consistent hydrogen

production with respect to wastewater strength, but did not

increase the hydrogen production. The bio-hydrogen gene-

rated from glucose as substrate resulted in a hydrogen

content of 48% [25], 62% [26], and 52 ~ 75% [21],

whereas sucrose yielded a hydrogen content of 35 [27] and

66% [28] while xylose yielded 32% of hydrogen [29]. The

anaerobic fermentation of jackfruit peel waste [30] and

palm oil mill effluent [31] resulted in a hydrogen content of

57 and 59% respectively.

The soluble COD removal percent during an influent TS

concentration of 5 and 7% are in shown in Fig. 8. The

increase in soluble COD removal percent confirmed the

digestion of substrate into end products into its gaseous

form. Moreover the accumulation of volatile fatty acid

content in the reactor when operated at 5 and 7% TS was

in the range between 462 and 580 mg/L which confirmed

the conversion of VFA into gaseous end product occurred

at steady phase without hindering the microbial reaction.

Lay et al. 2003 [32] stated that high solid organic waste

such as egg, lean meat, fat meat, chicken skin, potato, and

rice yielded a VFA content of 18, 35, 4, 11, 23, and 5 g/L

Fig. 5. Biogas generation versus operating period under continu-ous and intermittent power input of 2 and 2.5 V (influent TSconcentration of 2%).

Fig. 6. Hydrogen content in biogas versus operating period undercontinuous and intermittent power input of 2 and 2.5 V (influentVS concentration of 2%).

Fig. 7. Cumulative biogas generation versus operating period at anintermittent input voltage of 2 V and total solid of 5 and 7%.


as acetate at 6.25 d hydraulic retention time, when heat

shock digested sludge from pig manure was used as a

seeding material. Horiuchi et al. [33] stated an average

volatile fatty content of 3,500 mg/L during the anaerobic

acidogenesis at pH 5. Whereas Chang and Lin [23] stated

a VFA value ranging between 11,083 and 13,693 mg

COD/L at an hydraulic retention time of 4 ~ 24 h, while

treating synthetic substrate in upflow anaerobic sludge

blanket (UASB) reactor. In the case of sucrose as a sub-

strate during hydrogen fermentation resulted in a VFA

concentration of 3,505 mg/L Chen et al. [34] and 11,852

mg/L Lin and Lay [35] respectively.

As the digester pH was maintained in the acidic range

the chance of survival of methanogenic bacteria was remote,

which was well reflected in the absence of methane in the

generated biogas. The digester pH and redox potential was

found to be 5.5 ± 0.2 and −380 ± 17 mV in the present

investigation. The observed pH values were in corrobo-

ration with the earlier studies conducted by Ueno et al.

[36], Lin and Chang [37], Lay [38], and Hawkes et al. [39]

who confirmed that the acidogenic condition were more

favored for generating hydrogen when using consortium of

anaerobic bacteria. Liu and Shen [40] stated that the hydro-

gen yield increased for pH 5 ~ 8 while decreased at pH 9

during anaerobic digestion of starch. Lee et al. [41] find-

ings revealed that the fermentation of sucrose didn’t yield

hydrogen at pH 3, 11, and 12, however a low hydrogen

yield was observed at pH 5.0 ~ 5.5. Oh

et al. 2003 [21]

stated a pH value of 6.2 for maximum hydrogen produc-

tion, while Lin and Cheng [29], Khanal et al. [42] stated a

pH value of 5.5 ~ 6.0. Moreover at a digester pH below 4

the metabolic shift occurred from acid to solvent produc-

tion phase [43].

4. Conclusion

The foregoing study based on the anaerobic digestion and

in situ electrohydrolysis of dairy bio-sludge proved its

capability of digesting the organic matter coupled with

hydrogen generation. The volatile solids present in bio-

sludge were churned out by the microbes resulting in fatty

acids and hydrogen as gaseous end product. In order to

hasten the hydrogen yield the accumulated volatile fatty

acids were subject to electrohydrolysis, during this process

the proton released from fatty acids were combined with

electron to produce hydrogen at the cathode. The experi-

ments were conducted under continuous and intermittent

mode of input voltage ranging from 0.5 to 2.5 V for vary-

ing influent volatile solids concentration at a pH 5.3 ± 0.2.

The results favored intermittent mode of input voltage

rather than continuous supply. An influent total solids

concentration of 7% (64 and 120 mg/L VS) at an inter-

mittent input voltage of 2 V and a hydraulic retention time

of 15 days resulted in a volatile solids and soluble COD

removal efficiency of 83 and 74%, while the cumulative

gas generation was 1,051 L with a hydrogen content of

72%.

Acknowledgement

The authors wish to thank the Management of Prathyusha

Institute of Technology & Management for their financial

support of this work.

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