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Simultaneous biohydrogen production andwastewater treatment based on the selectiveenrichment of the fermentation ecosystem

Iulian Zoltan Boboescu a, Vasile Daniel Gherman a, Ion Mirel a,Bernadett Pap b, Roland Tengolics c, Gabor Rakhely c,d, Kornel L. Kovacs c,d,Eva Kondorosi d, Gergely Maroti a,d,*a “Politehnica” University of Timisoara, Hydrotechnical Engineering Dept., Timisoara, Romaniab Seqomics Biotechnology Ltd., Szeged, HungarycUniversity of Szeged, Dept. of Biotechnology, Szeged, HungarydHungarian Academy of Sciences, Biological Research Centre, Szeged, Hungary

a r t i c l e i n f o

Article history:

Received 30 January 2013

Accepted 30 August 2013

Available online xxx

Keywords:

Biohydrogen

Pretreatment

Microbial consortia

Synthetic wastewater

* Corresponding author. Hungarian Academy308270455.

E-mail addresses: [email protected] ([email protected] (B. Pap), [email protected]@gmail.com (E. Kondorosi),

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0360-3199/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.ijhydene.2013.08.1

a b s t r a c t

Biohydrogen production from synthetic wastewater as substrate was studied in anaerobic

small scale batch reactors. Enriched anaerobic mixed consortia sampled from various

environments were used as parent inocula to start the bioreactors. Selective enrichments

were achieved by various physical and chemical pretreatments and changes in the mi-

crobial communities were monitored by metagenomic and molecular diagnostics ap-

proaches. Experimental data showed the feasibility of biohydrogen production using

synthetic wastewater as substrate. The hydrogen generation capability of the different

mixed consortia is clearly dependent on the pretreatment methods. The described

approach opens the possibility for an alternative way towards simultaneous wastewater

treatment and renewable energy generation.

Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

1. Introduction fuel (142 kJ/g or 61,000 Btu/lb) and can be transported for do-

Energy is indispensable in all fields of life. The demand for the

energy is permanently growing, but the reserves of our pri-

mary energy-carriers will be depleted within a few decades

[1,2]. Novel safe energy carriers have to be introduced.

Hydrogen satisfies all the requirements for a clean, alternative

fuel producing only water as by-product upon combustion. It

has the highest energy content per unit weight of any known

of Sciences, Biological Re

Boboescu), vasile.ghermfreemail.hu (R. [email protected]

cu IZ, et al., Simultaneontation ecosystem, Inter

2013, Hydrogen Energy P39

mestic/industrial consumption through conventional means

[3e5]. In addition to this, H2 gas is safer to handle than do-

mestic natural gas. It can be used directly in the internal

combustion engines or in fuel cells to generate electricity. Its

use in fuel cells is inherently more efficient than the com-

bustion currently required for the conversion of other poten-

tial fuels to mechanical energy [6e8].

search Centre, Temesvari krt. 62., Szeged 6726, Hungary. Tel.: þ36

[email protected] (V.D. Gherman), [email protected] (I. Mirel), ber-cs), [email protected] (G. Rakhely), [email protected] (K.L. Kovacs),.hu (G. Maroti).

us biohydrogen production and wastewater treatment basednational Journal of Hydrogen Energy (2013), http://dx.doi.org/

ublications, LLC. Published by Elsevier Ltd. All rights reserved.

mailto:[email protected]










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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y x x x ( 2 0 1 3 ) 1e92

Among various hydrogen production processes, biological

ways are known to be the least energy intensive (direct

photolysis, indirect photolysis, photofermentation and dark

fermentation). Dark fermentation process can utilize various

organic wastes as substrate for fermentative hydrogen pro-

duction, thus it is considered a viable biohydrogen evolution

method driven by the anaerobic metabolism of the key in-

termediate, pyruvate. The complete oxidation of glucose

would yield a stoichiometry of 12moles H2 permole of glucose

but in this case no energy is utilized to support growth and

metabolism of the producing organism [9]. Dark H2 production

has the advantages of rapid hydrogen production rate and can

be operated at ambient temperature (30e40 �C) and pressure

[10,11], although under carefully chosen conditions thermo-

philes produce up to 60e80% of the theoretical maximum

demonstrating that higher hydrogen yields can be reached by

extremophiles rather than using mesophilic anaerobes [12].

Reducing the cost of wastewater treatment and finding

ways to produce useful products from wastewater has been

gaining importance in view of environmental sustainability.

One way to reduce the cost of wastewater treatment is to

simultaneously generate bioenergy by utilizing the organic

matter present in wastewater. Wastewaters generated by

various industrial processes are considered to be the ideal

substrates because they contain high levels of easily degrad-

able organic material. In the processes established so far,

organic pollutants and wastes are converted into methane.

Recently, emphasis started to shift to the development of

novel anaerobic processes aiming the conversion of organic

pollutants into hydrogen, instead of methane [13]. Thus, H2

production using wastewater as fermentative substrate with

simultaneous treatment of wastewater might be an effective

way of tapping clean energy from renewable source in a sus-

tainable approach [14].

Bacteria and other microbes capable of hydrogen produc-

tion widely exist in natural environments such as soil,

wastewater sludge, compost, etc. [15e17]. Thus, well selected

and concentrated derivatives of these sources can be used as

inoculum for fermentative hydrogen production. Dark

hydrogen production processes using mixed cultures are

more efficient than those using pure cultures, because the

formers represent more simple systems to operate and easier

to control, and may accept a broader source of feedstock [18].

However, in a fermentative hydrogen production process

using mixed cultures, the hydrogen produced by hydrogen-

evolving bacteria can be utilized by hydrogen-consuming

bacteria. Thus, restriction or termination of the methano-

genic process is crucial to render H2 to an end-product in the

metabolic flow [19]. There are pretreatment possibilities to

permit selective enrichment of specific groups of parent cul-

tures by inhibiting H2-consuming methanogenic bacteria

[14,20]. Pretreatment also prevents competitive growth and

co-existence of further H2-consuming bacteria [21]. The

enrichment methods reported for hydrogen-producing bac-

teria frommixed culturesmainly include heat-shock, acid and

base treatment, aeration, freezing and thawing, chloroform,

sodium-2-bromoethanesulfonate or 2-bromoethanesulfonic

acid and iodopropane treatments [19,20,22].

In the present study, a two-step biohydrogen production

process was investigated using different types of microbial

Please cite this article in press as: Boboescu IZ, et al., Simultaneoon the selective enrichment of the fermentation ecosystem, Inter10.1016/j.ijhydene.2013.08.139

communities as starting inocula. Prior to the inoculation, se-

lective enrichments of the bacterial populationswere achieved

by various physical and chemical pretreatments. In the first

experimental step, a glucose rich environment was applied,

while in the second experimental step, defined synthetic

wastewater was used as fermentation substrate. The model

system was investigated in anaerobic small scale batch re-

actors. Our aim was to determine the factors involved in the

desired shift from the traditional biogas forming communities

to an ecosystem favoring hydrogen evolution rather than

methane formation. In amore simpleway, thespecific goalwas

the elaboration of a method suitable for the selective elimina-

tion of methanogenic archaea, therefore suitable for simulta-

neous biohydrogen production and wastewater treatment.

2. Materials and methods

2.1. Seed inocula

Four different inocula were used during the experimental

setups. Samples were taken from: Timisoreana’s brewery

effluent (S1), Bocsa’s natural pool (S2), USAMVBT methane

producing bioreactor (S3) and Timisoara’s wastewater treat-

ment plant (S4).

2.2. Identifying the optimum pretreatment methods forthe inoculum, in relation to the substrate used

In order to enrich the hydrogen producing bacteria, four pre-

treatment methods plus a control, were used for each of the

inoculum. The batch experiments were performed in tripli-

cate. The following pretreatment methods were used: heating

of the inoculum at 70 �C for one hour, acid pretreatment

bringing the pH down to 3 for 24 h at room temperature using

1 N HCl, ultra-sonication of the samples for 30 min at a dis-

continues discharge of 24 KHz (0.5 s discharge followed by

0.5 s pause) and a combination of all of the pretreatments.

2.3. Design of synthetic wastewater

Designed synthetic wastewater (SW) [(mg/l) glucosed3700,

NH4Cld500, KH2PO4d250, K2HPO4d250, MgCl2�6H2Od300,

FeCl3d25, NiSO4d16, CoCl2d25, ZnCl2d11.5, CuCl2d10.5,

CaCl2d5 and MnCl2d15] was used as substrates for H2 pro-

duction. The pH was adjusted to 6 using 1 N HCl.

2.4. Enrichment of hydrogen producing bacterialconsortia

Enrichment of the sediment samples was done in DMI me-

dium following pretreatment. One liter of the DMI medium

contained 5240mg of NH4HCO3, 6720mg of NaHCO3, 125mg of

K2HPO4, 100 mg of MgCl2, 15 mg of MnSO4, 500 mg of Na2S,

10 mg of FeSO4, 10 mg of resazurin and 17,800 mg of carbon

source (glucose) as a substrate in 1 L distilled water. The en-

richments were conducted in 30 ml serum vial with 20 ml of

DMI medium and 4 ml of pretreated sediment samples as

inocula. The bottleswere cappedwith rubber septum stoppers

and aluminum rings and the medium in each bottle was




i n t e rn a t i o n a l j o u rn a l o f h y d r o g e n en e r g y x x x ( 2 0 1 3 ) 1e9 3

flushed after inoculation with oxygen-free nitrogen until

medium became completely anaerobic. Incubation was done

at 30 �C for a period of seven days.

2.5. Batch mode experiments using syntheticwastewater as a substrate

The pretreated and enriched anaerobicmixedmicroflorawere

used as inocula in small scale bioreactors (100 ml serum vials)

using synthetic wastewater as a substrate. After the enrich-

ment, 10 ml (20%) inoculum was used to inoculate 50 ml of

synthetic wastewater in 100 ml serum vials. The bottles were

capped with rubber septum stoppers and aluminum rings and

the medium in each bottle was flushed after inoculation with

oxygen-free nitrogen until the medium became completely

anaerobic. Incubationwas done at 30 �C for a period of 15 days.

2.6. Analytical methods

Bacterial cell mass in each individual culture was determined

by measuring optical absorbance (OD) with the Jenway 6320D

Spectrophotometer at 600 nm. pH measurements were per-

formed every 48 h using a Thermo Scientific Orion 3-star

benchtop pH meter.

Quantity and composition of headspace gas of the cultures

were directly measured by gas chromatography using an

Agilent Technologies 7890AGC system equippedwith thermal

conductivity detector and argon as a carrier gas. The tem-

peratures of the injector, detector and column were kept at

30 �C, 200 �C and 230 �C, respectively. HP PLOTQ column

(15 m � 530 mm � 40 mm) was used. Since the concentration

gradient of H2 gas can be formed in the headspace, gas sam-

ples (0.5 ml) was taken out after mixing of the headspace gas

by sparging several times with gas-tight syringe.

Metabolic products in liquid phase, remaining sugars in the

medium and ethanol productionwere analyzedwith a Hitachi

LaChrom Elite HPLC (High Performance Liquid Chromatog-

raphy) equipped with a L-2490 Refractive Index Detector

(temperature was set to 41 �C) and a L-2350 column thermo-

stat. L-2200 Autosampler was used for the injection of 40 ml

sample. For the separation of compounds a Transgenomic

ICSep COREGEL-64H (7.8 � 300 mm) organic acid analysis

column was used. The temperature of the column was set at

50 �C. The elution was performed by a 0.01 M H2SO4 solution

with the constant flow of 0.8 ml min�1.

2.7. Plating experiments

For the cell number determination in the complex samples

before and after the pretreatments, TGM medium (30 g/L

tryptone, 1 g/L yeast extract, 10 g/L glucose) solidified with

1.5% agar was used. Dilution series of samples were plated in

triplicates and plates were incubated at 30 �C both aerobically

and anaerobically.

2.8. Total DNA extraction from samples

DNA from the complex samples was extracted and purified

according to describedmethods with somemodifications [23].

Samples (0.5 g) were extractedwith 1.3ml of extraction buffer.


After proper mixing, 7 ml of proteinase K (20.2 mg/ml) was

added. After incubation, 160 ml of 20% SDS was added and

mixed by inversion for several times with further incubation

at 60 �C for 1 h with intermittent shaking after every 15 min.

The samples were centrifuged at 13,000 RPM for 5 min and the

supernatant was transferred into new eppendorf tubes. The

remaining soil pellets were treated three times with 400 ml of

extraction buffer, 60 ml of SDS (20%) and kept at 60 �C for

15 min with intermittent shaking after every 5 min. The su-

pernatants collected from all four extractions were mixed

with equal quantity of chloroform and isoamyl alcohol

(25:24:1). Aqueous layer was separated and precipitated with

0.7 volume of isopropanol. After centrifugation at 13,000 RPM

for 15 min, the pellet was washed with 70% ethanol, dried at

room temperature and was dissolved in TE (10 mM Tris Cl,

1 mM EDTA, pH 8.0).

2.9. Determination of community composition by 16SrRNA method

The rates of surviving microbes were determined for each

sample in response to each pretreatment method. Bacterial

and archaeal specific 16S rRNA (63F 50-CAGGCCTAACA-CATGCAAGTC-30 1542R 50-AAGGAGGTGATCCAGCCGCA-30

and UA571F 50-GCYTAAAGSRICCGTAGC-30 UA1204R 50-TTMGGGGCATRCIKACCT-30 respectively) analyses were per-

formed on both the colonies and the total DNA isolates to

determine the bacteria versus archaea ratio. The 16S rRNA

based molecular method has widely been used for taxonomic

profiling for decades. Capillary sequencing was used to

determine the sequence of the amplified 16S rRNA fragments.

Identification was achieved by homology search using BLAST

(http://blast.ncbi.nlm.nih.gov/).

2.10. Metagenomic characterization of the microbialcommunities

Total DNA of selected samples (S1 in all experimental phases)

were prepared for high-throughput next generation

sequencing analysis performed on Ion Torrent PGM platform

(Life Technologies). An average of 291.322 sequencing reads

were generated for each sample with a mean read length of

161 nucleotides. Bioinformatic analyses (taxonomic profiling,

assessment of metabolic potential) were conducted using the

public MG-RAST software package, which is a modified

version of RAST (Rapid Annotations based on Subsystem

Technology) [24].

3. Results

3.1. Assessment of the microbial communitycomposition

In order to design, create and continuously control amicrobial

consortium capable of efficient biohydrogen generation along

with wastewater treatment, different types of anaerobic eco-

systemse all rich in organicmatterewere sampled for parent

inocula. These parent cultures were as follows: activated

sludge from a wastewater treatment plant reactor, activated


http://blast.ncbi.nlm.nih.gov/




sludge from a wastewater pretreatment plant from food in-

dustry, sludge from a municipal sewage system and sludge

from watercourses heavily polluted by organic material.

These ecosystems have a high biodiversity and are composed

of naturally formed microflora suitable for biodegradation of

complex organic substrates. Prior to the inoculation of syn-

thetic wastewater, the parent cultures were sequentially

pretreated with heat, acid, ultrasonication and a combination

of these three methods in order to assess their efficiency to

selectively enrich the microbial communities. These pre-

treatments aimed the selective enrichment of the specific

groups of parent cultures by inhibiting H2-consuming meth-

anogenic bacteria.

The rates of surviving microbes were determined for each

sample in response to each pretreatment method. Plating

experiments followed by molecular identification were per-

formed under aerobic and anaerobic conditions. The combi-

nation of the three pretreatmentmethods and ultrasonication

alone resulted in the most dramatic decrease in total micro-

bial cell number as determined by counting colony forming

units before and after the pretreatments. Acid and heat

treatments showed a moderate killing efficiency compared to

ultrasonication. Plating approach can provide a raw estima-

tion of the efficiency of cell killing of the various pretreatment

methods, however only a small fraction of the total microbial

content of the samples can be assessed this way. In order to

get a more accurate insight into the community composition

upon various pretreatments, molecular and genomic identi-

fication approaches were performed using purified total DNA

samples as templates. Bacterial and archaeal specific 16S

rRNA analyses were performed on both the colonies and the

total DNA isolates to determine the ratio of bacteria, archaea

and eukaryotes. As an independent method, high-throughput

sequencing-based metagenomics was applied to obtain an

even more detailed view on the taxonomic profile of the

samples. Table 1 summarizes the effect of the pretreatment

methods as well as the control series on S1 samples by

showing the composition of the microbial communities at

different experimental stages.

The microbial populations of each inoculum used to start

up the bioreactors showed dramatic changes in response to

different pretreatment methods and different environmental

conditions. Themost striking alterations were observed in the

Archaea/Bacteria ratio (Table 1). Clear decrease in the archaeal

Table 1 e Microbial composition of S1 (sample 1) at different e

Inoculum

Initial microbial population of Sample 1

Control series of Sample 1 at the end of the enrichment phase

Heat pretreatment of Sample 1 at the end of the enrichment phase

Acid pretreatment of Sample 1 at the end of the enrichment phase

Ultrasonication pretreatment of Sample 1 at the end of the enrichment p

Control series of Sample 1 at the end of the wastewater phase

Heat pretreatment of Sample 1 at the end of the wastewater phase

Acid pretreatment of Sample 1 at the end of the wastewater phase

Ultrasonication pretreatment of Sample 1 at the end of the wastewater p


populations was observed in the cases of most of the applied

pretreatments used for S1, while the growing rate of the

Bacteria domain is evident also. A closer look at these do-

mains reveals that, even though the composition of the

Archaea domain remains relatively constant, the Bacteria

domain suffers significant changes in its composition and

structure. In the case of S1, a shift can be noticed in the bac-

terial composition from an initial community with relatively

high biodiversity to a clearly less complex ecosystem. The

starting S1 sample composed of Proteobacteria (55.24%), Fir-

micutes (19,12%), Bacteroidetes (14.98%) and other phyla

(10.66%) completely restructured in response to various

treatments followed by culturing in synthetic wastewater (e.g.

the final bacterial composition of the heat pretreated inoc-

ulum at the end of the wastewater phase was dominated by

Firmicutes (84.29%) and harbored strikingly decreased num-

ber of Proteobacteria (12.31%) and other phyla (3.4%)) (Fig. 1).

This community rearrangement was observed following all

pretreatments, even the control series exhibited this phe-

nomenon, although to a lesser extent.

3.2. Effects of various enrichment methods on themetabolism of selected microbial communities

As a result of the culture enrichment performed in DMI media

clear differenceswere observed in the hydrogen evolution rate

of the different inocula. S1 and S4 showed a generally higher

hydrogen production, H2 content of the biogas reaching a

maximum of 38% in S1 pretreated by ultrasonication and 42%

in S4 pretreated also by ultrasonication. However, S2 showed

a maximum of 17% H2 in the biogas when pretreated by heat,

and S3 showed a maximum of 14% H2 in the biogas when

pretreated by ultrasonication (Fig. 2).

The pretreatments used during the enrichment step

showed different effects on the hydrogen production rates of

the inocula used. Generally, the pretreated inocula produced

higher amount of hydrogen in comparison with the controls.

In most of the cases, the inocula pretreated by ultrasonication

showed the highest hydrogen production (a maximum of

5094.47 ml total H2 in the case of S4), while the inocula sub-

jected to acid pretreatment generally showed the lowest rate

of hydrogen generation (a maximum of 1093.590 ml H2 in S3).

Basic metabolites (glucose, ethanol, butyric acid) were

measured using HPLC at the end of the culture enrichment

xperimental stages.

Domains (%)

Archaea Bacteria Eukaryota

6.3 91.3 2.4

18.3 79 2.7

10.9 86.7 2.4

6.2 92.2 1.6

hase 2.4 95.7 1.9

2.8 95.2 2

2.1 96 1.9

1.5 97 1.5

hase 0.9 97.1 2




Fig. 1 e Rearrangement of the bacterial communities of S1 in the course of the experimental phases; Panel A e initial

bacterial composition; Panel B e bacterial composition following the enrichment phase (heat pretreatment); Panel C e

bacterial composition following the wastewater phase (heat pretreatment).


phase (Fig. 3). The initial concentration of the glucose was

17,800mg/l. The highest glucose consumption ratewas shown

in S1 and S4 pretreated by ultrasonication (with a remaining of

620 mg/l glucose for S1 and 950 mg/l glucose for S4, respec-

tively). In comparison, S2 and S3 have lower glucose con-

sumption rates (with a maximum of 11,000 mg/l glucose

remaining in S2 pretreated with acid, and 12,570 mg/l in S3

without pretreatment). Minor differences were detected in the

ethanol concentration of the enriched inocula regardless the

pretreatment methods applied (Fig. 3). The ethanol concen-

tration ranged between 2000 and 2500 mg/l in most of the

samples, only acid treated S1 and combined pretreated S4

showed significantly lower ethanol concentrations. There are

differences in the butyric acid levels between the 4 samples of

different origin. The butyric acid levels are also dependent on

the pretreatment methods (Fig. 3). The butyric acid concen-

trations are generally higher in S1 and S4 compared to S2 and

S3. The highest butyric acid concentrations in S1 and S4 are

2870 mg/l and 3210 mg/l, respectively. In both cases samples

were pretreated by ultrasonication. The highest butyric acid

concentrations in S2 and S3 are 640 mg/l and 550 mg/l. Heat

pretreatment was applied in the former and ultrasonication

was used in the latter case. The concentration of lactic acid,

acetic acid and propionic acid greatly differed according to the

Fig. 2 eHydrogen content of the produced biogas at the end

of the enrichment step; C e control, A e acid treatment, H e

heat treatment, U e ultrasonication treatment and HAU e

combination of all the treatments.


various inoculum types and pretreatments (Fig. 3). The high-

est acetic acid concentrations were measured in S1 and S4,

1730 mg/l and 1030 mg/l respectively, both observed in sam-

ples treated by ultrasonication. The highest lactic acid con-

centrations were measured in S4 with a maximum

concentration of 3550 mg/l in the untreated control sample.

The propionic acid concentrations were quite low in most of

the samples except in S2 treated by acid, where a concentra-

tion of 880 mg/l propionic acid was detected.

The pH values were recorded throughout the enrichment

experiments. The starting pH value was 8.5, a permanent

decrease in the pH was measured during the enrichment

phase regardless the pretreatment methods and various

inocula. However, slight differences could be observed in the

final pH value between the samples. In the case of S1 no dif-

ferenceswere recorded between differently treated samples, a

final pH value of 4.0 was recorded for each condition. The

combined pretreatment resulted in the most basic final pH

(5.4) in S2, all other conditions resulted in a final pH value

between 4.2 and 4.5 for S2. The highest variance in the final pH

values was observed in S3 depending on the pretreatment

methods (pH values of 4.5e6.1). Similarly to S1, S4 showed

only minor differences in the final pH, ranging from 3.7 to 4.0.

3.3. Fermentation of synthetic wastewater usingdifferentially enriched starters

Significant biohydrogen production during the wastewater

experimental phase using synthetic wastewater of defined

compositionwas shown only in S1 and S3with S1 evolving the

highest amount of hydrogen (Fig. 4). S2 and S4 were

completely devoid of hydrogen production in the wastewater

phase regardless the pretreatment methods applied in the

enrichment phase.

S1 pretreated by ultrasonication showed only about 9%

hydrogen of the total biogas in the wastewater phase,

althoughmuch higher ratio (38%) was achieved by this sample

and treatment in the enrichment phase (Fig. 2). The highest

amount of hydrogen produced by S1 was generated by the

control series with a 27% value of the total biogas. Interest-

ingly, the control series of S3 generated no hydrogen during




Fig. 3 e Concentration of selected metabolites at the end of the enrichment step; Panel A e S1 inoculum, Panel B e S2

inoculum, Panel C e S3 inoculum and Panel D e S4 inoculum; C e control, H e heat treatment, A e acid treatment, U e

ultrasonication treatment and HAU e combination of all the treatments.


the wastewater phase, only pretreated S3 variants evolved

hydrogen (5e13% of the total biogas) (Fig. 4).

Since S1 generated the highest amount of hydrogen in the

wastewater phase, we have investigated the fermentation

metabolites only in this experimental setup. The fermentative

microenvironments of S1 showed significant differences

regarding the producedmetabolites, depending on the applied

pretreatment methods (Fig. 5). The initial concentration of

glucose in the synthetic wastewater was 3700 mg/l. The

highest glucose consumption rate was found in the control

series (without any pretreatment) leading to complete deple-

tion of the glucose by the end of the experiment (Fig. 6). To the

contrary, using combined pretreatment on S1 resulted in the

lowest glucose consumption rate with a remaining of

2315 mg/l glucose by the end of the experiment. The ethanol

Fig. 4 eHydrogen content of the produced biogas at the end

of the synthetic wastewater degradation step; C e control,

A e acid treatment, H e heat treatment, U e ultrasonication

treatment and HAU e combination of all the treatments.


levels in the S1 microenvironments measured during the

wastewater phase reflect the fermentation differences

depending on various pretreatments (Fig. 5). The highest

ethanol concentration was measured in the control series

with a maximum value of 2680 mg/l (day 12 of the experi-

mental setup) (Fig. 6). The lowest ethanol concentration was

measured in the case of the combined pretreatment with a

maximum value of 1915 mg/l (day 5 of the experimental

setup). The butyric acid concentrations clearly differed be-

tween the control series and the pretreated S1 variants (Fig. 5).

The control series resulted in a maximum concentration of

1300 mg/l for the butyric acid by the end of the wastewater

phase. In contrast, the lowest concentration for the butyric

acid was foundwhen acid pretreatment was applied, reaching

a maximum concentration of 243 mg/l towards the end of the

experiments (Fig. 5).

The concentration of lactic acid, acetic acid and propionic

acid highly differed between the control series and the pre-

treated S1 experiments (Fig. 5). The lactic acid concentrations

are significant in all pretreated S1 cases, the highest values

were measured in the heat pretreated S1 series showing a

maximum concentration of 1833.5 mg/l. Interestingly, only

traces of lactic acid weremeasured in the control series with a

maximum concentration of 87.5 mg/l. Generally low levels of

acetic acid were detected in the wastewater phase, comparing

these low levels in response to different pretreatments, the

relative highest concentration was found in the untreated

control series with a maximum of 487.5 mg/l (Fig. 5). The

pretreated S1 series ranged between 187 and 260 mg/l acetic

acid concentrations. Propionic acid was found only in traces




Fig.5

eConce

ntrationofm

etabolitesin

S1atth

eendofth

esy

nth

eticwastewaterdegra

dationstep;C

eco

ntrol,H

eheattreatm

ent,A

eacidtreatm

ent,Ueultra

sonication

treatm

entandHAU

eco

mbin

ation

ofallth

etreatm

ents.



throughout the wastewater phase regardless the type of pre-

treatment including the control series.

No major variations were detected in the pH values of the

control and the pretreated series of S1, the values ranged be-

tween 3.5 and 3.8 towards the end of the wastewater

experiments.

4. Discussions and conclusions

Studies aiming the determination of optimal pretreatment

methods of complex microbial communities for achieving

efficient dark hydrogen fermentation are sporadic. Moreover

there is certain disagreement between the existing pretreat-

ment method recommendations for enriching hydrogen-

evolving bacterial communities based on mixed cultures of

natural origin. Possible reasons for this disagreement are the

differences among these studies in terms of origin of starter

inoculum used, differences in the specific conditions of each

pretreatment method and the various natures of substrates

applied.

Here we described a two-phase method for obtaining

enriched bacterial communities suitable for hydrogen evolu-

tion fromwastewater substrate. Themain goal was to develop

a mixed microbial consortium suitable for simultaneous bio-

hydrogen production and efficient wastewater treatment.

Synthetic wastewater of defined composition was used as

model system. The first step was the enrichment of various

complex mixed cultures of natural origin using organic rich

liquid medium and various physical and chemical pretreat-

ment methods. Several properties of these enrichment cul-

tures were recorded including hydrogen-evolving ability,

main metabolites and pH values along and as a result of this

fermentation step. The enriched microbial consortia were

used as inocula in the second step for hydrogen evolution

using synthetic wastewater. Again, levels of evolved

hydrogen, pH and various metabolites were assessed.

Therewere striking differences in the hydrogen production

rate of S1, S2, S3 and S4 inocula between the enrichment

phase and the wastewater phase. During the enrichment

phase all samples evolved hydrogen, while using synthetic

wastewater as a fermentation substrate, only S1 and S3

generated hydrogen. In addition to this, significant differences

in the hydrogen evolution were observed for samples of the

same origin between the two phases. As an example S1

showed the highest hydrogen production after ultra-

sonication in the enrichment phase, however, the enriched S1

culture showed only minor hydrogen production in the

wastewater phase. Interestingly, while the control series of S1

showed the lowest hydrogen production until day 4 of the

experiment during the enrichment step, the enriched S1

control generated the highest hydrogen level in the waste-

water phase. The highest hydrogen concentration during the

enrichment was observed in S4 when pretreated by ultra-

sonication. Interestingly this enriched consortium was not

able to generate any hydrogen from synthetic wastewater. As

expected, glucose consumption values were directly corre-

lated with hydrogen evolution rates in both the enrichment

and wastewater phases. The butyric and acetic acid concen-

trations seemed to be also directly correlated with hydrogen




Fig. 6 e pH values, H2 production and concentration of metabolites in the course of the synthetic wastewater degradation

step in the control series of S1; Panel A e pH values, Panel B e H2 production and Panel C e metabolites concentration.


production in both experimental phases. The highest con-

centrations of these metabolites were measured in samples

showing the highest hydrogen production, indicating that the

acetate and butyrate fermentations might be important

metabolic pathways operated by the hydrogen evolving pop-

ulations. Also as expected lower hydrogen concentrations

were associated with higher levels of propionate and reduced

end-products such as ethanol and lactic acid.

As expected, significant changes occurred in the microbial

communities during the experimental steps as it was shown

by 16S rRNA sequencing and preliminary metagenomic

characterization. A general and not surprising conclusion was

that all types of physical and chemical treatments resulted in

decreasing biodiversity. However, a number of observed

changes in the ecosystems were dependent on the applied

pretreatment methods. A clear decrease was observed in the

archaea/bacteria ratio in response to each treatment (the

strongest effect was observed when ultrasonication was

applied), and the increasing rate of bacteria was correlated to

the elevated hydrogen production rate of the ecosystems. An

obvious rearrangement was observed in the bacterial com-

munities during the experimental phases, the Firmicutes

phylum became highly dominant by the end of the experi-

ment, however in the starting substrates the Proteobacteria

phylum was the most abundant.

Explanations for the observed hydrogen evolution pattern

can be speculated in various ways. Since ultrasonication

pretreatment resulted in the highest hydrogen evolution rate

in three samples out of four in the enrichment phase, it might

be hypothesized that ultrasonication favored anaerobes able

to thrive in medium of high glucose concentration and to

break down glucose into pyruvate andNADH. The oxidation of

pyruvate into acetyl-CoA requires the reduction of a ferre-

doxin (Fd) by pyruvate-ferredoxin oxidoreductase, which is

then oxidized by a hydrogenase that regenerates oxidized Fd

and hydrogen gas. Another hypothesis is that ultrasonication

as a pretreatment method generally eliminates the highest

number of living microorganisms in the samples, thereby

most effectively kills hydrogen consuming microbes as well,

especially methanogenic archaea, which are known to be

highly sensitive organisms. The surviving populations was


shown to be composed of mainly bacteria, considering that

the bacteria/archaea ratio was clearly higher in ultrasonicated

samples compared to samples subjected to other pretreat-

ment methods. This latter hypothesis is also supported by the

fact, that the total number of surviving colonies was the

lowest in the samples subjected to ultrasonication. The re-

sults of the wastewater phase fermentation might also sup-

port this speculation. The wastewater substrate is devoid of

high concentrations of organic carbon sources, thereby the

less diverse microbial communities and the smaller absolute

number of living cells might have a lower chance to propagate

and survive as well as to produce hydrogen. The lowest

hydrogen production observed in the samples subjected to

ultrasonication either alone or in combination fits well into

this picture.

In the future, more detailed microbial community charac-

terization will be performed using metagenomic approaches

and comparative analysis of the ecosystems of each sample in

all phases (starting inocula enrichment phase, wastewater

phase) are planned to be provided.

Acknowledgments

This work was supported by the following international (EU)

and domestic (Romanian and Hungarian) fundings: “SYMBI-

OTICS” ERC AdG EU grant, “ALGOLABH” Baross Gabor Pro-

gramme OMFB-00356/2010 (NKTH, Hungary), the strategic

grant “POSDRU” 107/1.5/S/77265, inside POSDRU Romania

2007e2013 co-financed by the European Social Fund e

Investing in People and “BIOSIM” PN-II-PT-PCCA-2011-3.1-

1129 European Fund.

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