Novel dark fermentation involving bioaugmentation with constructed bacterial consortium for enhanced biohydrogen production from pretreated sewage sludge

international journal of hydrogen energy 34 (2009) 7489–7496

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Novel dark fermentation involving bioaugmentation with constructed bacterial consortium for enhanced biohydrogen production from pretreated sewage sludge Shireen Meher Kotay*, Debabrata Das Department of Biotechnology, Indian Institute of Technology, Kharagpur, India

article info

abstract

Article history:

The present study summarizes the observations on various nutrient and seed formulation

Received 7 January 2009

methods using sewage sludge that have been aimed at ameliorating the biohydrogen

Received in revised form

production potential. Pretreatment methods viz., acid/base treatment, heat treatment,

4 March 2009

sterilization, freezing–thawing, microwave, ultrasonication and chemical supplementation

Accepted 14 May 2009

were attempted on sludge. It was observed that pretreatment was essential not only to

Available online 26 June 2009

reduce the needless, competitive microbial load but also to improve the nutrient solublization of sludge. Heat treatment at 121  C for 20 min was found to be most effective in

Keywords:

reducing the microbial load by 98% and hydrolyzing the organic fraction of sludge.

Biohydrogen production

However, this pretreatment alone was either not sufficient or inconsistent in developing

Sewage sludge

a suitable microbial consortium for hydrogen production. Hydrogen yield was found to

Pretreatment

improve 1.5–4 times upon inoculation with H2-producing microorganisms. A defined

Microbial consortium

microbial consortium was developed consisting of three established bacteria viz., Enter-

Nutrient formulation

obacter cloacae IIT-BT 08, Citrobacter freundii IIT-BT L139 and Bacillus coagulans IIT-BT S1. Following pretreatments soluble proteins and lipids (the major component of the sludge) were also found to be consumed besides carbohydrates. This laid out the concurrent proteolytic/lipolytic ability of the developed H2-producing consortium. 1:1:1 v/v ratio of these bacteria in consortium was found to give the maximum yield of H2 from sludge, 39.15 ml H2/g CODreduced. 15%v/v dilution and supplementation with 0.5%w/v cane molasses prior to heat treatment was found to further improve the yield to 41.23 ml H2/g CODreduced. ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Biohydrogen holds the promise for a substantial contribution to the future renewable energy demands. It seems particularly suitable for relatively small-scale, decentralized systems, integrated with agricultural and industrial activities or waste processing facilities. Biohydrogen is considered as a vital

solution to a sustainable world power supply and is currently being seen as the versatile fuel of the future, with the potential to replace fossil fuels. It has the key prospective to become the ideal means among the range of renewable H2 production technologies presently existing [1]. Despite having higher evolution rate of hydrogen, the yield of hydrogen from fermentative process is lower than that

* Corresponding author. Tel.: þ91 3222283758; fax: þ91 3222278707. E-mail address: [email protected] (S.M. Kotay). 0360-3199/$ – see front matter ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.05.109

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achieved using other methods, and thus the process is not economically viable in its present form [2]. The pathways and experimental evidences reported thus far reveal that a maximum of 4 moles of hydrogen could be obtained from substrates such as glucose. By employing cheap renewable substrates the capital cost can be brought down by several hundred folds, surmounting the existing yield limitations of this process [1–3]. Sewage sludge among all the renewable biomass is abundantly rich in nutrients as well as microbial diversity making it most near ideal match for microbial H2 production. Sludge is by far the largest in volume amongst the by-products of wastewater treatments, and it’s processing and disposal is perhaps one of the most complex environmental problems facing the engineer in this field [4]. The currently examined technology facilitates dual benefits, bioremediation and clean gaseous energy recovery. The major reason why sewage sludge has been focused as source of inoculum is that it is bountiful with enteric bacteria, which are potential H2producers. Several pure cultures [5–7] and mixed microbial cultures [8–13] have been successfully enriched and tested for H2-producing potential. Nevertheless, the pursuit for ideal microbe(s) for H2 production has thrust the researchers to screen various sources. On the contrast, utilization of sludge as substrate for H2 production hasn’t received much attention than what it actually deserves. The feasibility studies on fermentative H2 production from sludge as demonstrated by few researchers [14–26] suggest that lower yield is the major limitation which precludes the technology from being commercialized. However, these studies were successful in concluding certain critical findings viz.; i) various pretreatments can have significant effect of on H2 production [19,20] ii) nutrient formulation is necessary for amelioration of H2 yield [21] iii) mixed cultures are more efficient than pure cultures with respect to H2 production from sewage sludge [14–17,22–25] and iv) sequential H2 and CH4 production system using mixed consortia can augment the total gaseous energy recovery [18,26]. The main groups of the organic solids discovered in sewage sludge are proteins, carbohydrates, fats and oils. But these organics are mostly complex and under-utilized; hence pretreatment becomes essential to render them suitable for H2 fermentation. A mixed microbial consortium can make the process further advantageous through its ability to convert such diverse organics present in sludge to biohydrogen. Another credible option reported is co-digestion of sludge with carbohydrate rich waste like food waste [27] or molasses [22]. Most studies on biological H2 production from sewage sludge have exploited pure microbial strains or enriched mixed microbial flora. The present study deals with the development of a constructed microbial consortium that is suitable for H2 production from sludge. A synchronous objective was to determine an optimized pretreatment of sewage sludge that can maximize the H2 yield. This effort is first of its kind to use a constructed consortium for H2 production from pretreated sewage sludge. It is expected to address the problems associated with sewage sludge disposal through simultaneous generation of clean gaseous energy in the form of H2.

2.

Materials and methods

2.1.

Microorganism and culture condition

Three established H2-producing bacteria used in the present study viz. Enterobacter cloacae IIT-BT 08, Citrobacter freundii IITBT L139 and Bacillus coagulans IIT-BT S1 were cultured on growth media, Nutrient broth (Himedia laboratories). The liquid cultures for inoculum were grown at 37  C in an incubator–shaker at 250 rpm until they reached a uniform cell concentration (OD600). The maintenance of stock-cultures, preparation of seed culture and production media composition were same as reported earlier [7,28].

2.2.

Pretreatment of sewage sludge

Seven different pretreatment methods viz., acid/base treatment, heat treatment, freezing–thawing, microwave, ultrasonication and chemical supplementation were attempted on sludge. Table 1. describes the details of each treatment. The conditions of each pretreatment were chosen as reported optimal in earlier studies [13,19,25,29].

2.3.

Microbial enumerations

Heterotrophic bacteria were enumerated by using Standard plate count method and Nutrient agar (Himedia Inc.). A dilution series (103–105) of each sample was prepared by serially transferring a 1 ml portion of sludge. Plates were incubated at 37  C for 5 days [30]. Total coliforms were enumerated by using a five-tube Most Probable Number (MPN) method. Lauryl tryptose broth was used in the presumptive and confirmed phase of multiple tube test [30]. All media ingredients were purchased from Himedia Laboratories.

2.4.

Hydrogen production in batch fermentors

Batch experiments were carried out in jacketed glass reactor (of working volume 500 ml) placed on a magnetic stirring platform using the free suspended cells. Defined medium, MYG (1%w/v Malt extract, 0.4%w/v yeast extract and 1%w/v glucose) with glucose as limiting substrate has been found to be most suitable for H2 production in earlier studies. Malt and yeast extract

Table 1 – Pretreatment methods used on sewage sludge. Treatment Acid Base Heat Freeze-thaw Microwave Ultrasonication

pH 3–4 for 24 h using 0.1 N HCl at 25  C pH 10–11 for 24 h using 4 N NaOH at 25  C 121  C for 20 min 20  C/25  C for 6 h each in two cycles 600 W for 2 min (Samsung 3D shower wave, India) 20 kHz frequency, 140 W (1.4 W/ml) at 2 mm depth at 25  C (Ultrasonic cell disruptor XL, Misonix Inc.) Chemical supplementation 1%v/v CHCl3 at 25  C for 24 h CHCl3 BESA 10 mM at 25  C for 24 h

international journal of hydrogen energy 34 (2009) 7489–7496

ensured sufficient supply of nitrogen, phosphorus and growth factors for the bacteria. The pretreatment of sludge included sterilization, 15%v/v dilution and supplementation with 0.5%w/ v glucose. All the media and glassware were autoclaved for 15 min at 121  C and 15 lb pressure. 12 h old (OD600 ¼ 0.8) culture of the bacterium grown in nutrient broth (Himedia Inc.) was used as inoculum (10%v/v). Anaerobicity was maintained by flushing with Ar for 5 min after the inoculation.

2.5.

Methods of analysis

2.5.1.

H2

The gas was collected in a graduated water displacement system containing saline solution. The composition of the gas (in the collector) was analyzed by a Gas chromatograph (Perkin–Elmer) with TCD using Porapak-Q column and N2 as carrier gas at 5.62 kg/cm2 pressure and 20 ml/min flow rate. Injector, oven and detector temperatures were set at 120  C, 120  C and 200  C respectively. The chromatogram was developed and analyzed using software, Turbochrome Navigator (version 4.1) Perkin–Elmer Coorp.

2.5.2.

Carbohydrate/protein/lipid concentrations

Carbohydrate was measured by the phenol sulfuric acid method using glucose as the standard [31] while, protein was determined by the Lowry method using bovine albumin as the standard [32]. Lipid concentration was determined using two techniques. First, fatty solids concentration was measured by extraction by hexane according to Bridoux et al. [33]. Samples were acidified in order to maintain fatty acids in the nondissociated form. Hexane was added to samples which were agitated; fatty acids transferred from sludge to hexane fraction. Then the hexane phase was collected and evaporated (Rotavapor, Buchi Inc.). By weighting the extracted compounds, and by knowing the initial volume of sludge, it was possible to determine fatty solids concentration. The error due to this measure was around 15%.

2.5.4.

PCR-DGGE

The bacterial community of sludge before and after pretreatment was analyzed by DGGE of the PCR-amplified 16S rDNA. Primer 518-r and 357-f (with 40 bp GC clamp at the 50 -end) were used to amplify the approx. 200-bp fragment of the V3 region. The PCR programme corresponded to 20 cycles of three steps: 94  C for 1 min, 65  C for 0$75 min and 72  C for 1 min, 10 cycles of three steps: 94  C for 1 min, 55  C for 0$75 min and 72  C for 1 min followed by a final step at 72  C for 10 min. PCR products were stored at 4  C and analyzed on 1.5% agarose before DGGE. DGGE analysis of the amplicons obtained from PCR was performed using the Dcode Universal Mutation Detection system (Bio-Rad, Hercules, CA, USA) with 8% (v/v) polyacrylamide gels and a denaturant gradient of 30–60%. A 100% denaturing solution was defined as 7 mol/L urea and 40% formamide. Electrophoresis was performed for 16 h at 70 V in a 0.5  TAE buffer at 60  C. DGGE gels were stained with SYBR Green for 15 min and analyzed on GelDoc XR 1708170 system (Bio-Rad). The nucleotide sequences of the distinct bands from the DGGE were determined by sequencing and subsequently identified based on the sequence similarity (Fig. 2).

Physico-chemical characteristics

The effluent from the reactor was collected at regular interval of time for analysis. Physical parameters viz. COD, TS, VSS etc. were estimated using standard methods [30].

2.5.3.

2.5.6.

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3.

Results and discussion

3.1. Effect of various pretreatments on sludge characteristics 3.1.1.

Effect on sludge microbiology

The primary purpose of any pretreatment of sewage sludge is to enhance the efficiency of its anaerobic digestion. This is achieved through two mechanisms: i) reduction in the competitive microbial load and ii) increase in the organic availability. Table 2. enlists the characteristics of sludge before any pretreatment. Sewage sludge is abundant with enteric bacteria, some of which are known as potential H2-producers.

VFA and alcohols

VFA and alcohols present in the spent media were estimated by GC using FID Detector, capillary column (0.5 mm diameter  30 m length) at preset injector, oven and detector temperatures, 250  C, 200  C and 280  C respectively. The operation of the capillary column was amenable to a temperature programming process within 100–200  C (Flow rate of carrier gas (N2), combustion gas (H2) and zero-air were set at 20 ml/min, 50 ml/min and 500 ml/min respectively).

2.5.5.

Protease activity

The protease activity of 1.5 ml sludge suspension or supernatant (collected by centrifugation at 3000  g for 10 min) of sludge was measured according to Kim’s methods [34]. One unit of the enzyme activity was defined as the amount of the enzyme which degraded 1 mg azocasein in 60 min at 28 or 60  C.

Fig. 1 – Effect of various pretreatments on microbial load of sludge.

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Table 2 – Characteristics of raw sewage sludge before pretreatment. Parameter pH T.S. (g/l) T.S.S. (g/l) T.V.S. (g/l) T.D.S. (g/l) C.O.D. (g/l) B.O.D5 (g/l) T.O.C (% w/v) Total carbohydrate (% COD) Total protein (% COD) Total Lipid (% COD) C/N Ratio Standard plate count (CFU/g sludge) Total Coliform (CFU/g sludge)

Value 6.8 120.5 74.5 54.1 46.0 104.0 60.8 52.1 14.7 41.4 25.3 5.0 5.84  109 1.92  109

CFU ¼ colony forming units.

However, it also consists of several non-H2-producing microorganisms which can add to the nutrient competition. Further, sludge harbors methanogens and other H2-cosuming bacteria. Therefore, it is essential to eliminate these microbial populations in order to augment the H2 production. In the present study it was observed that heat treatment was most efficient in reducing the microbial load (Fig. 1). Microwave, ultrasonication and chemical supplementation had comparable and significant effect on microbial population (c.a. 80–90% reduction). Acid, base and freeze–thawing were

found to be comparatively less efficient for the purpose. Thermal treatment has been used widely employed for reduction of microbial load [13–15,22,25,29] and enrich H2producers. It is further advantageous because it can assist in sludge solubilisation [13,29]. To enhance anaerobic digestion of sludge it is necessary that microorganisms utilize maximum possible organics present in it. In other words the rate-limiting step of this biological process is organic matter hydrolysis.

3.1.2.

Microbial succession analyzed by DGGE

The presence of band corresponding to B. coagulans in the DGGE profile of sludge after all the pretreatments clearly implies the ability of the strain to withstand harsh conditions (Fig. 2). Thermoanerobacter sp. and Alicyclobacillus acidocalvarius were also detected after thermal pretreatment. Acidophiles like Acetivibrio cellulolyticus and A. acidocalvarius were also found to withstand acid pretreatment. Consistent presence of B. coagulans in call cases also indicates its dominance in the sludge. Although DGGE is not a quantitative method due to the bias introduced by PCR, the density change of each band can be explained as a consequence of a change in the relative abundance of the microbes in the microbial community [35]. It has already been established that B. coagulans isolated from sludge has significant H2-producing ability [7].

3.1.3.

Effect on sludge solubilisation

Solubilisation refers to describe the transfer from the particular fraction to the supernatant of centrifugation. In the present study, effect of various pretreatments on solubilisation of COD, proteins and carbohydrates was studied. COD solubilisation was found significant only in case of thermal and microwave pretreatment (Fig. 3), which means it is solely linked to heat. This was further justified by decrease in the VSS/TSS ratio achieved in case of heat treatment (data not shown). Similar findings were reported recently during the studies on the effect of thermal treatment of sludge [36]. With an intention to improve H2 production ability of sludge, a few studies have been reported [13–15,22,25,29]. Among the organic solids, carbohydrates are most degraded components during anaerobic digestion. However, it has been hypothesized that protein and lipids also contribute to H2 production to certain extent. After the pretreatment, proteins were noticed to be better solubilised than carbohydrates or lipids (Fig. 3). In accord to previous findings, heat pretreatment was the only method which rendered considerable solubilisation of organics. No solubilisation of organics was observed in case of freeze-thawing and chemical supplementation (Fig. 3).

3.2. Effect of various pretreatments on H2-producing mixed microbial culture enrichment

Fig. 2 – Microbial profile (as revealed by DGGE) of sludge before and after pretreatment.

With an objective of enriching mixed cultures with H2 production potential, several pretreatment methods have been attempted on sludge [14–17,23–25]. In the present study however, the pretreatments alone were not sufficient enough to augment the H2 yield from sludge. Though most pretreatments were able control CH4 production, only heat treatment

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Fig. 3 – Effect of various pretreatments on sludge solubilisation.

to a considerable extent was able to improve the H2 production (Fig. 4).

3.3.

Construction of defined consortium

Due to the insignificant improvement in H2 yield after pretreatments, inoculation with pre-established H2producing microorganisms was attempted. A defined microbial consortium consisting of three established H2-producing bacteria viz., E. cloacae IIT-BT 08, C. freundii IIT-BT L139 and B. coagulans IIT-BT S1 was developed exclusively for this purpose. The three stains individually have been investigated in earlier studies and established as potential H2-producers [7,22,28].

Table 3 – Substrate degradability of the strains. Enzyme

E. cloacae IIT-BT 08

C. freundii IIT-BT L139

B. coagulans IIT-BT S1

Amylase Cellulase Xylanase Lipase Protease

þ þ þ þ 

þ   þ 

þ þ þ þ þ

Fig. 4 – Effect of various pretreatments on hydrogen and methane production from sludge.

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Table 4 – Effect of various substrates on H2 production using various inocula. H2 yield (mol H2/mol substrate)

Carbon source

Glucose Cellobiose L-Arabinose Fructose Maltose Potato starch C.M. cellulose D-Xylose Sucrose

H2 content (%v/v)

E. cloacae

C. freundii

B. coagulans

E. cloacae

C. freundii

B. coagulans

2.8 5.4 1.5 1.6 1.4 – – 0.95 6.0

2.4 0 0.2 1.4 1.4 – – 0 5.4

2.28 5.6 1.9 1.6 1.4 – – 1.2 5.3

60  5 53  5 51  5 55  5 60  5 48  5 45  5 40  5 62  5

54  5 – 45  5 51  5 54  5 40  5 – – 55  5

50  5 50  5 45  5 49  5 49  5 43  5 40  5 50  5 50  5

Following inoculation, the H2 yield from sludge was found to improve 1.5–4 times. Besides H2 production ability, these bacteria also shared morphological (small rods) and physiological (facultative anaerobes) similarities. They have also been found to co-exist in natural environments like sewage sludge. As pure cultures the strains were already reported to convert complex substrates to H2 [7,28]. In the present study they were assessed further for their ability to degrade various organics to H2. Among the three strains, B. coagulans was found to be most diverse with respect to organic substrate degradability. However, E. cloacae was better in terms of converting various carbohydrates to H2. Besides H2, E. cloacae has also been

reported to produce amylase [37]. Similarly in earlier studies, C. freundii has been established for alkaline lipase production [38]. In different reports, it has been found that B. coagulans had cellulase [39], xylanase [40], amylase [41] and protease [34,42] producing abilities (Table 3). These evidences on versatile metabolic characteristics of the bacteria may be the possible reasons for the increased COD reduction and H2 yield obtained using the consortium. The three strains when considered in consortium were catering better substrate utilization spectrum, overall COD reduction and H2 yield (Table 4). Their ability to co-exist and produce H2 however needs to further investigated for any possible symbiotic associations.

Fig. 5 – Profiles of cell mass and substrate concentration for various consortium ratios.

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also to improve the nutrient solublisation of sludge. Thermal treatment was most effective in reducing the microbial load and hydrolyzing the organic fraction of sludge. However, the pretreatments alone were either insufficient or inconsistent in developing a suitable microbial consortium for H2 production. A defined microbial consortium developed was found to improve H2 yield by 1.5–4 times. The concurrent proteolytic/ lipolytic abilities of the developed consortium were responsible for better substrate conversion and higher H2 production. 1:1:1 v/v ratio of these bacteria in consortium was found to give the maximum yield of hydrogen from sludge.

Acknowledgements We gratefully acknowledge the financial support extended by Department of Biotechnology, Govt. of India and Ministry of New and Renewable Energy, Govt. of India in the form of a research grant. We also wish to acknowledge Department of Environmental Engineering, Technical University of Denmark, Lyngby, Denmark for their infrastructural support. Fig. 6 – Hydrogen production by various consortium ratios using MYG and sludge.

3.4. Optimization of constructed consortium concentration suitable for H2 production This study has been a unique attempt to realize the H2 production ability of a constructed microbial consortium consisting of three bacteria derived from different sources. In the preliminary studies, their H2 production ability was tested on a defined medium, MYG (1%w/v Malt extract, 0.4%w/v yeast extract and 1%w/v glucose) with glucose as limiting substrate. MYG has been found in earlier studies to be more suitable for H2 production. Optimal conditions of pH, temperature, inoculum age and inoculum volume 6, 37  C, 12 h and 10%v/v respectively were considered as reported in previous studies [7,28]. Four different ratios (1:1:1, 2:1:1, 1:2:1 and 1:1:2) of bacteria in consortium were attempted. 1:1:1 v/v was found most suitable for augmentation of H2 production. Fig. 5. depicts the cell mass and substrate utilization profiles of each of the consortium ratios tried. 1:1:1 v/v ratio of these bacteria in consortium was found to give the maximum H2 yield using both defined medium and sludge (Fig. 6.). The H2 yields of 360.39 ml H2/g CODreduced and 39.15 ml H2/g CODreduced achieved were higher than those reported earlier. 15% v/v dilution and supplementation with 0.5%w/v cane molasses prior to heat treatment was found to further improve the yield to 41.23 ml H2/g CODreduced.

4.

Conclusions

The present study was an attempt to improve the biohydrogen production potential using various nutrient and seed formulation methods on sewage sludge. Pretreatment was essential not only to reduce the needless, competitive microbial load but

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