Microbial community structure of anaerobic sludge for hydrogen production underdifferent acid pretreatment conditionsJingjing Wan, Yanyan Ning, Xianyang Shi, Dawei Jin, Shouqin Li, and Yongchun Chen

Citation: Journal of Renewable and Sustainable Energy 5, 023126 (2013); doi: 10.1063/1.4800199 View online: http://dx.doi.org/10.1063/1.4800199 View Table of Contents: http://scitation.aip.org/content/aip/journal/jrse/5/2?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Fermentative hydrogen production by newly isolated Clostridium perfringens ATCC 13124 J. Renewable Sustainable Energy 6, 013130 (2014); 10.1063/1.4863085 Analysis of microbial diversity and optimal conditions for enhanced biogas production from swine wasteanaerobic digestion J. Renewable Sustainable Energy 5, 053143 (2013); 10.1063/1.4822256 Biogas production from broiler manure, wastewater treatment plant sludge, and greenhouse waste by anaerobicco-digestion J. Renewable Sustainable Energy 5, 043126 (2013); 10.1063/1.4818771 Experimental study on rheological characteristics of high solid content sludge and it is mesophilic anaerobicdigestion J. Renewable Sustainable Energy 5, 043117 (2013); 10.1063/1.4816814 Anaerobic fermentation hydrogen production from apple residue: Effects of sludge pretreatments J. Renewable Sustainable Energy 4, 013104 (2012); 10.1063/1.3682077

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Microbial community structure of anaerobic sludgefor hydrogen production under different acid pretreatmentconditions

Jingjing Wan,1 Yanyan Ning,1 Xianyang Shi,1,a) Dawei Jin,1 Shouqin Li,2

and Yongchun Chen2

1College of Resources and Environmental Engineering, Anhui University, Hefei 230039,China2Lab for Ecological environment, Huainan Mining Group, Huannai 232001, China

(Received 10 October 2012; accepted 22 March 2013; published online 3 April 2013)

The effect of different pretreatment methods on anaerobic H2 production from a

sucrose-rich synthetic wastewater was investigated in this work. The substrate

utilization, formation of aqueous products, H2 production and microbial diversity

in anaerobic sludge all markedly depended on these pretreatment methods. The

highest H2 production according to the values for the maximum H2 production rate

of 27.57 ml h�1, specific H2 production rate of 2.962 ml g-VSS h�1, and H2 yield

of 1.617 mol H2 mol glucose�1 was observed in the H2 production by the sludge

pretreated with butyrate (pH¼ 2). Correspondingly, the main microbial

communities were Clostridium sp. HPB-16, Clostridium sp. HPB-46, Clostridiumsp. HPB-2, Clostridium sp. HPB-4, Oxalobacteraceae bacterium QD1, uncultured

bacterium clone HPR93, uncultured Olsenella sp. clone J27, and uncultured

bacterium clone SR_FBR_E5. This result demonstrates that acid pretreatment such

as butyrate could be also as an effective method to enrich H2-producing bacteria

from anaerobic sludge for large-scale biological H2 production.

[http://dx.doi.org/10.1063/1.4800199]

I. INTRODUCTION

Biological H2 production by fermentative bacteria has some advantages such as high H2

evolution rate, applicability to different types of wastes, and feasibility for industrialization.1

One of the difficulties is how to prepare lots of anaerobic H2 producing inocula economically

and easily from natural sources.2 To improve H2 production, the inocula must be pretreated to

eliminate H2-consuming bacteria (HCB) such as methanogens, homoacetogens, and sulfate

reducing bacteria for enriching H2-producing bacteria (HPB).3

To date, several pretreatment approaches, mainly including heat-shock, acid, base, aeration,

freezing, and thawing, chloroform, and sodium 2-bromoethanesulfonate (BESA), have been

used and compared to enrich HPB in previous studies, but some conclusions are conflicting.4,5

Heat pretreatment of inocula represses not only non-spore forming HBC, e.g., methanogens, but

also non-spore forming HPB.6 The utilization of BESA was not suitable for large scale applica-

tions of biological H2 production due to economic reasons. In another research, the chloroform

treatment has been found to be the most effective pretreatment compared with heat and acid

pretreatment.7 Cheong et al.8 demonstrated that an acid pretreatment significantly promoted H2

production by sludge, while base pretreatment was found the best in other researches.6 A com-

bination of different pretreatments may be more effective to eliminate of H2 consumers.9

The reason for the disagreement on the best pretreatment method to enrich HPB was that

different inoculum sources and substrates were used in different studies.10 Thus, it is important

to evaluate the effectiveness of a pretreatment method on inocula because the different

a)Author to whom correspondence should be addressed. Electronic mail: shixi381@163.com.

1941-7012/2013/5(2)/023126/8/$30.00 5, 023126-1

JOURNAL OF RENEWABLE AND SUSTAINABLE ENERGY 5, 023126 (2013)

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inoculum sources had their specific bacterial structures and fermentation patterns.5,11 Compared

with other pretreatments, acid pretreatment gained more attention because it is simple and

effective for enriching HPB from anaerobic sludge.12 So far, acid pretreatment was usually

conducted with hydrochloric acid.4,5,8 Little information was available to investigate the effec-

tiveness using the main aqueous products of fermentative H2 production (other acids), such as

acetate and butyrate, to select HPB. Furthermore, in order to evaluate the effectiveness of a pre-

treatment method on inocula, investigation into microbial community structures becomes

crucial.

Therefore, the purpose of this work was to explore the feasibility of applying two organic

acids, i.e., acetate and butyrate, to enrich HPB from anaerobic sludge. In addition, the relation-

ship between the metabolites, microbial community, and H2 production at each acid pretreat-

ment was also investigated.

II. MATERIALS AND METHODS

A. Seed sludge and pretreatment

The seed sludge was collected from a full-scale upflow anaerobic sludge blanket reactor

treating citrate-producing wastewater. Prior to use, it was first washed with tap water five times

and was then sieved with a sieve of 60 Mesh. For the acid-pretreatment, the sludge was

adjusted to acidic pH 2, 3, and 4 with 1 N acetate and butyrate, respectively, for 24 h, and was

then adjusted back to pH 7.0 with the addition of 1 N NaOH. In this work, the seed sludge was

also heated at 102 �C for 90 min in the heat pretreatment for assessing the effects of acid pre-

treatment on biological H2 production. The sludge without any pretreatment was used as a

control.

B. Batch experiment

The batch experiment for H2 production was conducted in 600 ml glass reactors with tripli-

cate. Seven sludge seeds with pretreatment (10 ml) having volatile suspended solids (VSS) con-

centration of 19.2 g l�1 and nutrient solution of 10 ml were added to each glass reactor, respec-

tively. The total working volume of each reactor was filled to 300 ml with distilled water. The

nutrient solution contained (mg l�1) sucrose 10000; NH4HCO3 2024; K2HPO4�3H2O 800;

CaCl2 50; MgCl2�6H2O 100; FeCl2 25; NaCl 10; CoCl2�6H2O 5; MnCl2�4H2O 5; AlCl3 2.5;

(NH4)6Mo7O24 15; H3BO4 5; NiCl2�6H2O 5; CuCl2�5H2O 5; and ZnCl2 5. The pH of the mixed

liquor in each reactor was adjusted by 1 mol l�1 HCl or NaOH. Prior to operation, each reactor

was flushed with argon for 5 min to ensure anaerobic condition. The cultures were placed in

air-bath shaker at 36 6 1 �C with 120 rpm. The volume and composition of biogas and concen-

tration of the soluble metabolites were measured until the H2 production ceased in each reactor.

C. Chemical analysis

The amount of biogas production was determined daily using water-replace equipment. The

percentage of H2 in the gas was analyzed by using a gas chromatography (GC) (Model 6890A;

Lunan Co., China) equipped with a thermal conductivity detector and a 2 m stainless column

packed with 5 A molecular sieve. The concentrations of ethanol and volatile fatty acids (VFAs)

were determined by the second GC (Model 6890NT, Agilent Inc.) equipped with a flame ioni-

zation detector and a 30 m� 0.25 mm� 0.25 lm fused-silica capillary column (DB-FFAP). The

liquor samples were first centrifuged at 12 000 rpm for 5 min, and were then acidified with for-

mic acid. Thereafter, they were filtrated through a 0.2 lm membrane and were finally measured

for free acids. The sucrose concentration was measured using anthrone-sulfuric acid method.13

The VSS was measured according to the Standard Methods.14

The cumulative H2 production data were analyzed by applying a modified-Gompertz

equation,15

023126-2 Wan et al. J. Renewable Sustainable Energy 5, 023126 (2013)

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H ¼ P � exp �expRm � e

Pðk� tÞ þ 1

� �� �; (1)

where H is the accumulative H2 production (ml) during the fermentation time t (h), P is the H2

production potential (ml), Rm is the maximum production rate (ml h�1), k is the lag-phase time

(h), and e is 2.718281828. The values of P, Rm, and k were nonlinearly evaluated using the

function of software Origin 6.1.

D. Microbial analysis

For microbial analysis, the sample numbers were recorded as H (heat), A2 (acetate pH¼ 2),

A3 (acetate pH¼ 3), A4 (acetate pH¼ 4), B2 (butyrate pH¼ 2), B3 (butyrate pH¼ 3), B4 (butyr-

ate pH¼ 4), Y (seed sludge), and Y1 (control, using seed sludge to perform H2 production test),

respectively. The total genomic community DNA of sludge samples was extracted and purified.16

The product from DNA extraction of each sample was then amplified using two successive PCRs

[nested Polymerase chain reaction (PCR)] with the primer For/Dev. A 1500-bp fragment of

the 16 S rRNA gene including the V3 region was first amplified in a reaction mixture with a

final volume of 50 ll that contained 2 ll of DNA template, 2 ll of primer for (50-GAGTTTGATCCTGGCTCAG-30), 2 ll of primer Dev (50-AGAAAGGAGGTGATCCAGCC-30),1 ll of each dNTP, 5 ll of PCR buffer, 0.5 ll (0.5 U) of Taq enzyme and sterile water to a final

volume of 50 ll. The amplification program was 94 �C for 2 min; 30 cycles of 94 �C for 1 min,

65 �C for 45 s, and, finally, 72 �C for 5 min.

Second, the 550-bp fragment of V3-V5 region was amplified using the primers F357 (50-CCTACGGGAGGCAGCAG-30) and R907 (50-CCGTCAATTCCTTTGAGTTT-30). Amplification

was performed in a total reaction volume of 50 ll as described above. The amplification program

was 94 �C for 5 min; 20 cycles of 94 �C for 1 min, 65 �C-55 �C for 1 min, 72 �C for 1 min, 10 cycles

of 94 �C for 1 min, 55 �C for 1 min, and, finally, 72 �C for 8 min. The PCR products were examined

with 1% (W/V) agarose gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) was conducted using the D-code system (Bio-

Rad, USA). The PCR products were loaded onto 6% (w/v) polyacrylamide gel with 45%-65% dena-

turant gradients. Electrophoresis was performed with a constant voltage of 100 V at 60 �C for 17 h.

After electrophoresis, the gels were stained with nucleic acid dyes for 30 min, and then washed by

hydrogen peroxide for 20 min, finally photographed with a gel photo system (Tanon 2000, China).

The Shannon-Weiner diversity index (H) was utilized to indicate the diversity of the bacteria com-

munity. The index, H, was calculated on the basis of the following equation:17

H ¼ �Xs

i¼1

pi ln pi; (2)

where pi is the proportion of the ith band intensity in total intensity of the gel lane and S is the

number in the gel lane. The cluster analysis of bacteria community was carried out using an

unweighted pair group method with arithmetic average (UPGMA).

The selected DGGE bands were cut from the gel and eluted in 30 ll Tris-EDTA (TE) at

4 �C overnight for identifying the microbial populations. After the DNA template of the bands

was re-amplified, the products were purified using a PCR purification Kit (Sangon, Shanghai,

China), ligated into pEASY-T1 vector, and transformed into competent Escherichia coli based

on the manufacturer’s instructions. The positive clones were screened with an appropriate

media supplemented with ampicillin and the V3-16 S rDNA sequences were then submitted to

the GenBank database. The homology with other 16 S rDNA sequences in the database was

determined by the BLAST software.

III. RESULTS AND DISCUSSION

A. Substrate utilization

Figure 1 illustrated the degradation consumption of sucrose with acid- and heat treated

sludge as well as control. Sucrose concentration was decreased rapidly in the initial 100 h of all

023126-3 Wan et al. J. Renewable Sustainable Energy 5, 023126 (2013)

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tests. About more than 60% of sucrose was utilized at the cease of H2 production. The anaero-

bic sludge treated by butyrate at pH of 2 gave the maximum substrate degradation efficiency of

93.67%, much higher than those of the control test and other pretreatments. These results

showed that substrate degradation efficiencies ranged from 60.38% to 93.67% were influenced

by the different pretreatment methods.

B. Formation of aqueous products

In the anaerobic fermentation of sucrose, VFAs and alcohols were simultaneously produced

as a useful indicator for monitoring H2 formation. Table I summarized the effects of different

pretreatment methods on the VFAs production. According to Table I, acetate and butyrate, to-

gether accounting for above 88% in total VFAs, were found to be the dominant aqueous prod-

ucts in all tests. By contrast, ethanol and propionate were also present, but in relatively low

quantities. The maximum acetate production of 1239.55 mg l�1 was achieved at butyrate pre-

treatment with pH of 2. A high acetate concentration was a benefit to produce H2 by a combi-

nation of photosynthetic bacteria with anaerobic acidogenic bacteria from wastewater.18

Notably, heat pretreatment gave the highest butyrate production of 3165.91 mg l�1 which was

not readily utilized by Rhodobactercapsulatus for H2 generation.18 Such an aqueous product

distribution indicates that butyrate-type fermentation was present in this experiment.2

C. Analysis of H2 production

To examine the influence of different pretreatments on H2 production, the experimental

data for cumulative H2 were fitted to Eq. (1) and the results were given in Table II. The

FIG. 1. The substrate degradation curve at various pretreatments.

TABLE I. Distribution of aqueous products at various pretreatments.

Pretreatment methods Ethanol (mg l�1) Acetate (mg l�1) Propionate (mg l�1) Butyrate (mg l�1) Total (mg l�1)

Control 87.35 6 3.08 634.90 6 12.21 101.58 6 2.73 1255.00 6 21.08 2078.83 6 55.44

Heat 352.27 6 6.34 467.67 6 5.32 129.22 6 3.32 3165.91 6 72.63 4115.07 6 85.83

Acetate (pH¼ 2) 75.91 6 1.46 606.93 6 8.44 25.51 6 0.96 1478.75 6 32.34 2187.10 6 42.97

Acetate (pH¼ 3) 56.05 6 0.98 470.74 6 5.21 18.16 6 0.46 959.90 6 18.82 1504.85 6 37.85

Acetate (pH¼ 4) 71.94 6 2.52 554.49 6 7.56 64.92 6 1.25 1115.85 6 32.56 1807.20 6 52.56

Butyrate (pH¼ 2) 140.06 6 3.32 1239.55 6 42.56 28.75 6 0.46 2833.91 6 63.47 4242.27 6 76.42

Butyrate (pH¼ 3) 125.87 6 1.74 845.89 6 8.23 36.66 6 1.82 1887.47 6 43.56 2895.89 6 46.63

Butyrate (pH¼ 4) 92.63 6 1.37 218.62 6 3.47 36.26 6 1.33 1431.23 6 33.64 1778.74 6 33.67

023126-4 Wan et al. J. Renewable Sustainable Energy 5, 023126 (2013)

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determination of coefficient (R2) of all non-linear analysis exceeded 0.99, suggesting that the

modified-Gompertz model was able to adequately describe the progress of cumulative H2 pro-

duction in this experiment.15 Accordingly, the specific H2 production rate and H2 yield were

calculated based on sludge concentration and substrate concentration, respectively. The amounts

of evolved H2 and lag time were strongly dependent on the different pretreatment methods. The

shortest lag time was 2.54 h with acetate (pH¼ 4) pretreatment sludge. However, the highest H2

production by virtue of the values for the maximum H2 production rate of 27.57 ml h�1, spe-

cific H2 production rate of 2.962 ml g-VSS�h�1, and H2 yield of 1.617 mol H2 mol glucose�1

was observed for the sludge pretreated with butyrate (pH¼ 2), which is higher than those of

with heat pretreatment and control. Moreover, the heat pretreatment is regarded as hard to

implement and not cost-effective (e.g., requirement for thermal energy). Many non-spore HPB

might be destroyed, resulting in relatively lower H2 production efficiency similar to that of con-

trol in this work. On the other hand, the H2 yield was found higher in this work than in previ-

ous study with HCl to pretreat anaerobic sludge.12 Thus butyrate pretreatment (pH¼ 2) could

be as a simple, economic, and effective approach to enrich HPB from anaerobic sludge com-

pared with heat pretreatment. These results also demonstrate that selection of the best inoculum

for H2 production was vital to improve H2 yield of anaerobic fermentative H2 generation.

D. Microbial diversity

The index of H indicating the diversity of the bacteria community was determined based

on Eq. (2) (Table III). The results showed that each sludge sample had substantial richness

and well-diversified bacterial communities. The lowest microbial diversity (H¼ 2.3579) was

observed in sludge with heat pretreatment since many non-spore bacteria were destroyed.

Thus, the limitation of such a pretreatment resulted in reduction of microbial community.

The H of seed sludge (3.1642) was higher than those of other samples, suggesting that the

microbial diversity in anaerobic sludge were markedly affected by different pretreatment

methods.

Figure 2 illustrated that the sludge samples were separated by DGGE approach. To identify

the microbial species, 16 S rDNA fragments extracted from the prominent DGGE bands were

re-amplified, purified, cloned to pEASY-T3 vector, and sequenced. The results showed that dif-

ferent pretreatments led to significant microbial population shift from the seed sludge. In these

sequences, 12 bacteria clones, respectively, belonged to Clostridia, Sporolactobacillaceae,

Burkholderiales, and Coriobacteridae (Fig. 3). Bands 1, 4, 5, 9, and 12, respectively, related to

Clostridium sp. HPB-16, Clostridium sp. HPB-2, Clostridium sp. HPB-4, Oxalobacteraceaebacterium QD1, and uncultured bacterium clone SR_FBR_E5 were found to exist in all sam-

ples, implying that these bacteria could not be inhibited under acid and heat conditions.

Clostridium sp. HPB-16, Clostridium sp. HPB-2, and Clostridium sp. HPB-4 were closely

related to H2 production, indicating that they could be HPB.19,20 Bands 2 and 8 were enriched

TABLE II. Kinetic parameters of H2 production for various pretreatment methods.

Pretreatment

method K (h) Rm (ml h�1) P (ml)

Specific H2 production

rate (ml g-VSS�h�1)

H2 yield (mol H2

mol glucose�1) R2

Control 2.623 6 0.123 14.18 6 1.25 327.3 6 9.9 1.524 6 0.209 0.833 6 0.033 0.9950

Heat 3.277 6 0.254 21.6 6 1.96 326.0 6 12.2 2.321 6 0.209 0.830 6 0.0311 0.9982

Acetate (pH¼ 2) 4.151 6 0.372 21.75 6 2.24 564.1 6 11.1 2.33 6 0.209 1.436 6 0.0281 0.9942

Acetate (pH¼ 3) 2.887 6 0.265 11.94 6 0.93 369.1 6 10.2 1.283 6 0.209 0.939 6 0.0259 0.9901

Acetate (pH¼ 4) 2.544 6 0.107 20.36 6 1.85 416.3 6 11.3 2.188 6 0.209 1.060 6 0.0287 0.9953

Butyrate (pH¼ 2) 3.991 6 0.228 27.57 6 2.37 635.4 6 11.2 2.962 6 0.209 1.617 6 0.0284 0.9947

Butyrate (pH¼ 3) 3.231 6 0.283 20.59 6 1.87 536.1 6 10.3 2.212 6 0.209 1.364 6 0.0272 0.9919

Butyrate (pH¼ 4) 2.723 6 0.249 16.07 6 1.46 304.3 6 8.5 1.727 6 0.209 0.775 6 0.0217 0.9956

023126-5 Wan et al. J. Renewable Sustainable Energy 5, 023126 (2013)

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from the seed sludge and were identified as Clostridium sp. HPB-46 and Sporolactobacillus sp.

QC81-06, respectively. Bands 3 and 7, respectively, affiliated to C. Tyrobutyricum and

Clostridium sp. HPB-26, as well as Bands 6, 10, and 11, all related to uncultured only appeared

in some samples.

The above results demonstrated that employing different pretreatment methods resulted in

the change of microbial community structure for H2 production. In particular, the compositions

of dominant microbial community are directly responsible for H2 generation. A lower pH was

benefit to suppress HCB such as methanogens and enrich HPB including Clostridia.21 Thus,

among these pretreatments, the butyrate (pH¼ 2) pretreatment was found the effective way to

enrich HPB. Anaerobic sludge pretreated with butyrate (pH¼ 2) shows the highest H2 produc-

tion. Correspondingly, the main microbial communities were Clostridium sp. HPB-16,

Clostridium sp. HPB-46, Clostridium sp. HPB-2, Clostridium sp. HPB-4, Oxalobacteraceaebacterium QD1, uncultured bacterium clone HPR93, uncultured Olsenella sp. clone J27, and

uncultured bacterium clone SR_FBR_E5. This suggests that such a pretreatment could be also

as an effective method to screen HPB from anaerobic sludge.

TABLE III. Shannon-Wiener index (H), evenness (E), and richness (S) of bacterial popularity.

Pretreatment

methods

Seed

sludge

Acetate

(pH¼ 2)

Acetate

(pH¼ 3)

Acetate

(pH¼ 4) Control

Butyrate

(pH¼ 2)

Butyrate

(pH¼ 3)

Butyrate

(pH¼ 4) Heat

H 3.1642 2.8029 2.7845 2.8974 2.8308 2.5841 2.8487 2.5264 2.3579

S 25 17 17 19 18 14 18 13 11

FIG. 2. DGGE profiles of sludge samples with various pretreatments.

023126-6 Wan et al. J. Renewable Sustainable Energy 5, 023126 (2013)

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IV. CONCLUSIONS

The main aqueous products of fermentative H2 production such as acetate and butyrate

could be as a simple, economic, and effective approach to enrich HPB from anaerobic sludge

compared with heat retreatment. The substrate degradation efficiencies ranged from 60.38% to

93.67% were influenced by the different pretreatment methods. The highest H2 production by

virtue of the values for the maximum H2 production rate of 27.57 ml h�1, specific H2 produc-

tion rate of 2.962 ml g-VSS�h�1, and H2 yield of 1.617 mol H2 mol glucose�1 was observed for

the sludge pretreated with butyrate (pH¼ 2). The formation of aqueous products, H2 production

and microbial diversity in anaerobic sludge all markedly depended on different pretreatment

methods. DGGE analysis demonstrated that acid pretreatment showed a desirable selectivity on

inhibition of HCB rather than HPB.

FIG. 3. Phylogenetic tree of dominant microbial species based on 16S rDNA. sequence from different sludge samples:

phylogenetic tree was constructed using Neighbor-joining algorithm.

023126-7 Wan et al. J. Renewable Sustainable Energy 5, 023126 (2013)

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ACKNOWLEDGMENTS

The authors wish to thank the Natural Science Key Foundation of the Anhui Higher Education

Institutions (KJ2011A003), the Natural Science Foundation of Anhui province (1208085ME61),

the scientific research project of Huainan mining group (HNKY- JT-JS-2012-3), the professional

project of Characteristics of Anhui province (2011-8), the Natural Science Foundation of China

(51278001), and the NSFC–JST Joint Project No. (20610002) for the support of this study.

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