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Journal of Biotechnology 155 (2011) 387– 395

Contents lists available at ScienceDirect

Journal of Biotechnology

jou rn al hom epage: www.elsev ier .com/ locate / jb io tec

nfluence of carbohydrates and proteins concentration on fermentative hydrogenroduction using canteen based waste under acidophilic microenvironment

. Venkateswar Reddy, K. Chandrasekhar, S. Venkata Mohan ∗

ioengineering and Environmental Centre (BEEC), Indian Institute of Chemical Technology (IICT), Tarnaka, Hyderabad 500 607, India

r t i c l e i n f o

rticle history:eceived 3 May 2011eceived in revised form 29 June 2011ccepted 21 July 2011vailable online 30 July 2011

eywords:naerobic sequencing batch reactor

AnSBR)

a b s t r a c t

Functional role of biomolecules viz., carbohydrates and proteins on acidogenic biohydrogen (H2) pro-duction was studied through the treatment of canteen based composite food waste. The performancewas evaluated in an anaerobic sequencing batch reactor (AnSBR) at pH 6 with five variable organicloading conditions (OLR1, 0.854; OLR2, 1.69; OLR3, 3.38; OLR4, 6.54 and OLR5, 9.85 kg COD/m3-day).Experimental data depicted the feasibility of H2 production from the stabilization of food waste and wasfound to depend on the substrate load. Among the five loading conditions studied, OLR4 documentedmaximum H2 production (69.95 mmol), while higher substrate degradation (3.99 kg COD/m3-day) wasobserved with OLR5. Specific hydrogen yield (SHY) vary with the removal of different biomolecules and

astewater treatmentolatile fatty acids (VFA)uffering capacityurface methodology

was found to decrease with increase in the OLR. Maximum SHY was observed with hexose removal atOLR1 (139.24 mol/kg HexoseR at 24 h), followed by pentoses (OLR1, 108.26 mol/kg PentoseR at 48 h), pro-teins (OLR1, 109.71 mol/kg ProteinR at 48 h) and total carbohydrates (OLR1, 58.31 mol/kg CHOR at 24 h).Proteins present in wastewater helped to maintain the buffering capacity but also enhanced the H2 pro-duction by supplying readily available organic nitrogen to the consortia. Along with carbohydrates andproteins, total solids also registered good removal.

© 2011 Elsevier B.V. All rights reserved.

. Introduction

Generation and disposal of wastes is an integral part of society.xploitation of wastes as substrate for biohydrogen (H2) produc-ion with simultaneous treatment is considered as one of thettractive and effective way of tapping energy from renewableesources in a sustainable approach (Vijayaraghavan et al., 2007;hairattanamanokorn et al., 2009; Chong et al., 2009; Venkataohan, 2009). This facilitates dual benefits to the environment

uch as renewable energy generation and waste stabilizationBenemann, 1996; Hawkes et al., 2002; Van Ginkel et al., 2005;ovacs et al., 2006; Venkata Mohan et al., 2008c, 2010; Wangnd Wan, 2009; Wu et al., 2009; Venkata Mohan et al., 2010).ecently, fermentative H2 production from a variety of substrates,aste materials and wastewaters using mixed cultures has been

eported (Fan et al., 2006; Venkata Mohan et al., 2007a,b, 2008a,b;hang et al., 2007; Prasertsan et al., 2009; Venkata Mohan, 2009;uo et al., 2010; Mohanakrishna et al., 2010). Among all known

eed stocks, canteen waste is considered as one of the richestources of organic material with composite nature. Presence ofigh content of volatile solids and moisture is its key characteristic

∗ Corresponding author. Tel.: +91 40 27191664; fax: +91 40 27191664.E-mail address: vmohan s@yahoo.com (S.V. Mohan).

168-1656/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.jbiotec.2011.07.030

feature and the composition is highly variable depending on theirsources. High organic fraction of food waste and its conversion toenergy or harness valuable products with simultaneous treatmentmakes the process economically viable. Anaerobic fermentationis a preferred method for resource recovery from canteen waste,because it has several advantages, including volume reduction,waste stabilization, and biogas recovery (Cho et al., 1995; Limet al., 2000). Han and Shin (2004) reported a two stage processfor converting food waste to H2 and methane. Zhu et al. (2008)studied the influence of co-digestion of food waste with sludgeon H2 production in batch scale operation and attributed the pro-cess improvement due to increase in buffering capacity of theco-digestion mixture. Major criteria for the selection of canteenwaste as suitable substrate for H2 production was its easy availabil-ity, cost and feasibility, higher carbohydrate, protein content andeasily biodegradable nature. However, its origin, composite nature,protein and oil content of waste and particles size variation makethe treatment process difficult. Degradability of each componentof canteen waste is affected by the environmental conditions. Car-bohydrates are easily degradable and on hydrolysis they producesimple sugars like hexoses, pentoses and soluble acid metabolites.

Hexoses are used for the production of H2 and energy requiredfor the maintenance of normal metabolic activities occur in theorganisms. Pentoses release the electron carrying molecules whichare involved in H2 production and production of nucleic acids which

3 Biotech

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88 M.V. Reddy et al. / Journal of

re used in building of genetic material in the organism. During H2roduction, proteins serve as the nitrogen source to improve therowth and activity of acidogenic H2 producing bacteria (Ding et al.,008). Previous studies reported the importance and influence ofrotein in maintaining the system buffering capacity during H2roduction. Bai et al. (2004) observed H2 production from glucosend peptone as substrates at different concentrations to investi-ate the role of carbohydrate and protein present in the artificialastewater and reported that suitable ratios of glucose and pep-

one enhanced the growth of H2 producing bacteria. Therefore, theain objective of this study is to assess the influence of different

iomolecules viz., carbohydrates and proteins present in the can-een based composite food waste on acidogenic H2 production innaerobic sequencing batch reactor (AnSBR). The performance ofhe bioreactor was evaluated with respect to the removal of chem-cal oxygen demand (COD), carbohydrates, proteins and total solidsTS) along with their H2 conversion efficiency at variable substrateoading conditions.

. Experimental methodology

.1. Canteen waste

Food waste collected from Indian institute of chemical tech-ology (IICT) canteen was used as substrate in the present study.he institute caters the need of about 700–1000 people per day.aste generated was composite in nature including uneaten

ood and food preparation leftovers mostly comprising of boiledice (60 ± 5%; wet weight basis) followed by cooked vegetables14 ± 4%), un-cooked vegetables (spoiled) (2 ± 1%), cooking oil6 ± 2%), vegetable peelings (3 ± 2%), cooked meat (4 ± 2%), cookedsh (2 ± 1%) and boiled spices (1.5 ± 1%). The water content of wastearied between 6 and 10%. Prior to feeding, the collected canteenaste was masticated using electrical blender and filtered through

tainless steel sieve to remove coarse materials so as to avoid poten-ial clogging problems. Oil fraction of waste was separated throughravity separator. The resulted oil free filtrate was used as a feedfter adjusting it to the required organic loading rate (OLR) by dilut-ng with domestic sewage. Supplementation with domestic sewagepH, 7.5; COD, 420 mg/l; total dissolved solids (TDS), 460 mg/l) sup-orts the adequate amount of pH buffer, minerals and nutrient

alance of wastewater. The characteristics of the waste prior toubstrate load adjustment were summarized in Table 1. Wastewa-er showed the presence of high concentration of organic load withood biodegradability (BOD/COD ∼ 0.75).

able 1hysicochemical characteristics of canteen based composite food waste.

Characteristics Values

pH 6.52 ± 1.2Colora 70,000 ± 120Turbidityb 7710 ± 80TSS 23,068 ± 74TS 36,290 ± 110Total alkalinity 1084 ± 20TDS 13,220 ± 75Chlorides 1420 ± 104Volatile fatty acids (VFA) 5879 ± 340Total chemical oxygen demand (COD) 330,000 ± 560Biochemical oxygen demand (BOD5) 247,500 ± 640Nitrates 9.8 ± 0.42Proteins 31,250 ± 35Total carbohydrates 95,874 ± 92Hexoses 54,578 ± 156Pentoses 40,624 ± 144Oil and grease 75,000 ± 130

a Units in Hazen units.b Units in NTU; all other parameters were expressed in mg/l.

nology 155 (2011) 387– 395

2.2. Inoculum

Anaerobic mixed consortia from full scale UASB reactor wasused as parent inoculum (Venkata Mohan et al., 2008a). Priorto inoculation, the parent culture was washed in saline buffer(5000 rpm, 20 ◦C) and enriched in designed synthetic wastewater(DSW) [glucose – 3 g/l; NH4Cl – 0.5 g/l, KH2PO4 – 0.25 g/l, K2HPO4– 0.25 g/l, MgCl2 – 0.3 g/l, CoCl2 – 25 mg/l, ZnCl2 – 11.5 mg/l, CuCl2– 10.5 mg/l, CaCl2 – 5 mg/l, MnCl2 – 15 mg/l, FeCl3 – 25 mg/l, NiSO4– 16 mg/l, COD – 3600 mg/l] under anaerobic microenvironment atpH 6.0 (80 rpm; 48 h).

2.3. Reactor configuration and operation

Bench scale anaerobic sequencing batch reactor (AnSBR)with suspended growth configuration was fabricated with atotal/working volume of 1.0/0.82 l and a gas holding capacity of180 ml was used in this study. The bioreactor was made leakproof with proper inlet and outlet arrangements. The biore-actor was operated in sequencing/periodic discontinuous batchmode with a total cycle period of 72 h [hydraulic retentiontime (HRT)] consisting of 15 min of FILL, 71 h of REACT (anaer-obic), 30 min of SETTLE and 15 min of DECANT phases. At thebeginning of each cycle, immediately after withdrawal, a pre-defined volume (0.75 l) was fed to the reactor during FILLphase. The reactor was kept in suspension during the REACTphase. The bioreactor was operated at variable substrate load-ing conditions of wastewater viz., OLR1 (0.85 kg COD/m3-day),OLR2 (1.69 kg COD/m3-day), OLR3 (3.38 kg COD/m3-day), OLR4(6.54 kg COD/m3-day) and OLR5 (9.85 kg COD/m3-day). Prior tofeeding wastewater pH was adjusted to 6.0 using 3 N NaOH/88%ortho-phosphoric acid. Constant COD removal efficiency andH2 production were considered as the indicators to assessthe stabilized performance of the bioreactor prior to changingfeed load. Reactor was operated at ambient room conditions(29 ± 2◦C).

2.4. Analyses

H2 stored in the head space of the bioreactor was estimatedusing a microprocessor based pre-calibrated sensor (FMK satel-lite 4–20 mA version, ATMI GmbH Inc., Germany). The system wascalibrated once in every two days using the calibration cap pro-vided with the instrument, and the sensor had a measuring rangeof 0.01–10% H2 with 5 s response time in a temperature rangeof 20–80 ◦C. Total alkalinity, pH, TS, volatile fatty acids (VFA) andCOD (closed refluxing method) were determined according to thestandard methods (APHA, 1988). Total and individual carbohy-drates (hexoses and pentoses) were analyzed by phenol–sulfuricacid method (Rao and Pattabiraman, 1989). Total proteins wereanalyzed by colorimetric method (Lowry et al., 1951).

3. Results and discussion

3.1. Biohydrogen

Experimental data depicted the feasibility of fermentative H2production through the stabilization of composite canteen basedfood waste. However, the H2 production and substrate degrada-tion patterns were found to depend on the substrate concentration(Fig. 1a). H2 production rate differed significantly with varying OLR.H2 production was observed to start from 4th h of operation irre-

spective of the OLR applied. Among the five loading rates studied,OLR4 showed maximum H2 production rate (1.52 mmol/h at 36 h)followed by OLR3 (1.13 mmol/h at 28 h), OLR2 (1.04 mmol/h at24 h), OLR5 (0.99 mmol/h at 44 h) and OLR1 (0.85 mmol/h at 20 h).

M.V. Reddy et al. / Journal of Biotech

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)

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Fig. 1. Performance of bioreactor with respect to: (a) hydrogen production rate; (b)c

Tiirttdltclow5satineiltrawo

Among the amino acids, l-glutamic acid can produce H2, while thepresence of l-proline will enhance H production. Protein based

TC

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umulative hydrogen production.

he time interval at which maximum production rate observedncreased with increase in OLR. This is an indication for the lagn the metabolic activities at initial time with higher OLR and theequirement of additional time for the conversion of organic mat-er into H2. Irrespective of the OLR, production rate was observedo drop gradually after attaining a maximum value. However, therop was higher in the case of lower OLR near 72 h, while it was

ower in the case of higher OLR studied. This can be attributedo the availability of substrate even at 72 h also at higher loadingonditions (OLR4 and OLR5). Contrary to the production rate, cumu-ative H2 production (CHP) showed an increased trend till the endf the experimental run (Fig. 1b). CHP was observed to increaseith increase in OLR (OLR1, 36.45 mmol; OLR2, 42.90 mmol; OLR3,

2.46 mmol) up to OLR4 (69.94 mmol), while OLR5 (42.05 mmol)howed lower CHP than other OLR in the initial phase and showed

sharp increment in the later phase of operation. CHP was observedo stabilize after 48 h in the case of OLR1, while in the case of OLR2t was noticed after 52 h and at 64 h with OLR3. OLR4 and OLR5 didot showed any stability more over showed an increasing trendven in the later phase of operation till 72 h. However, incrementn CHP was sharp in the case of OLR5 compared to OLR4 in theater phase of operation which might be attributed to the adapta-ion of the consortia for higher loading conditions. Few of previouseports documented H2 production using food waste as substratend mixed culture as biocatalyst. Anaerobic co-digestion of food

aste with sewage sludge documented specific H2 production rate

f 111.2 ml H2/g VSS/h (Kim et al., 2004). Shin et al. (2003) reported

able 2onsolidated results pertaining to substrate degradation and H2 production during exper

OLR (kg COD/m3-day) CHOR (%) HexoseR (%) PentoseR (%) ProteinR (%)

0.854 73.64 71.52 72.16 84.53

1.690 64.72 61.65 58.23 84.90

3.380 56.55 53.83 53.71 78.42

6.540 51.41 50.24 49.74 81.23

9.850 34.71 34.06 33.56 67.18

HOR – carbohydrates removal; HexoseR – hexose removal; PentoseR – pentose removal;hydrogen production rate.

nology 155 (2011) 387– 395 389

specific H2 production rate of 22.6 ml H2/g VSS/h using food wasteas substrate.

3.2. Carbohydrate hydrolysis

Total as well as individual carbohydrates were analyzed alongwith COD to understand the consumption of carbohydrates dur-ing H2 production. Canteen based food waste used in this studyis enriched with organic acids, sugar-based compounds, aminoacids and fatty acids. The biodegradability of canteen waste ismainly related to carbohydrate material, which is the main precur-sor for H2 production. Initial concentration of total carbohydrateswas observed to increase with increasing OLR and removal wasgradually increased from 0 h to 72 h in all the cases (Fig. 2a).Among the five OLR studied, OLR1 showed maximum carbohy-drate removal efficiency (73.64%) followed by OLR2 (64.72%), OLR3(56.55%), OLR4 (51.41%) and OLR5 (34.71%). Contrary to removalefficiency, carbohydrate removal was observed to be higher athigher loading conditions [OLR5, 3627 mg/l; OLR4, 3898 mg/l;OLR3, 2228 mg/l; OLR2, 1432 mg/l and OLR1, 896 mg/l]. Hexosesand pentoses showed almost similar pattern as carbohydratesdegradation with respect to time and OLR (Fig. 2b and 2c). Com-pared to pentoses, hexoses showed slight higher degradation forall the OLR except OLR1. OLR1 showed highest removal efficiency[71.52%, hexoses; 72.16%, pentoses followed by OLR2 (61.65%, hex-oses; 58.23%, pentoses), OLR3 (53.83%, hexoses; 53.71%, pentoses),OLR4 (50.24%, hexoses; 49.74%, pentoses) and OLR5 (34.06%, hex-oses; 33.56%, pentoses)] (Table 2).

3.3. Proteins hydrolysis

Proteins and amino acids after anaerobic degradation produceorganic nitrogen which is used for the microbial growth and main-tenance. Generally proteins form good fraction of any food wastebut vary depending on its source. Total proteins at OLR1 wereobserved to be 485 mg/l initially and decreased to 75 mg/l by theend of the cycle period (Fig. 2d). Likewise at all the other OLR,protein concentrations showed reduction during H2 production(OLR2: 828 mg/l and 125 mg/l; OLR3: 1502 mg/l and 324 mg/l;OLR4: 2814 mg/l and 528 mg/l; OLR5: 3578 mg/l and 1174 mg/l)indicating their role in the fermentation process. Optimal H2 pro-duction and cell growth were reported earlier with the combinationof carbohydrates and protein source during the fermentation pro-cess. High protein content could enhance the H2 production bydirect organic nitrogen supply to microorganisms (Gabrielyan et al.,2010). Proteins generally get degraded into amino acids by theaction of proteolytic enzymes or get denatured into simple struc-tures by acid hydrolysis. Amino acids get converted to differentorganic acids by transamination and deamination reactions andproduce ammonia along with H2. Ammonia is a chief source fornitrogen which is principally required for normal cell activities.

2organic nitrogen is transformed into inorganic nitrogen in the formof ammonia in anaerobic microenvironment. Ammonia and amino

imental study.

CODR (%) TSR (%) HPR (mmol/h) CHP (mmol) SHY (mol/kg CODR)

74.91 65.54 0.852 (20 h) 36.45 16.4871.61 52.62 1.045 (24 h) 42.90 14.6963.39 50.87 1.133 (28 h) 52.46 8.3957.36 47.67 1.521 (36 h) 69.94 5.4940.56 39.87 0.991 (44 h) 42.05 4.56

ProteinR – protein removal; CODR – COD removal; TSR – total solids removal; HPR

390 M.V. Reddy et al. / Journal of Biotechnology 155 (2011) 387– 395

0 12 24 36 48 60 720

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b

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d

e

toses

gi

R

fitb

Fig. 2. Changes in concentration of: (a) total carbohydrates; (b) hexoses; (c) pen

roups released from proteins maintain suitable pH by the alkalin-ty produced (Ding et al., 2008).

–NH2 → NH4+ + HCO3

− + CO2 + H2O (1)

The potential decrease in pH caused by VFA generated during

ermentation was buffered by the bicarbonate produced dur-ng protein degradation (Eq. (1)) (Ding et al., 2008). As long ashe biodegradation of protein accompanies the overall organiciodegradation, the pH could be stabilized at a certain level. The

Time (h)

; (d) proteins; (e) total solids with the function of different organic loading rate.

observed H2 production for extended periods of time might be dueto the presence of proteins and their degradation to ammonia alongwith bicarbonate production. Optimized ratio of carbohydrates andproteins yield higher H2 production, especially in the case of solidwaste like kitchen based and canteen based food waste. Proteins

present in the canteen waste not only provided buffering capacityto neutralize the VFA but also enhanced the H2 production by pro-viding readily available organic nitrogen such as soluble proteinsand amino acids to the microorganisms.

iotechnology 155 (2011) 387– 395 391

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M.V. Reddy et al. / Journal of B

.4. Total substrate degradation

Experimental data evidenced good substrate (COD) degrada-ion in combination with H2 production (Fig. 3). This indicateshe function of canteen waste as good carbon source for metabol-cally harness H2. COD removal efficiency was especially highert lower OLR. On the contrary, substrate degradation rate (SDR)as observed to increase with increase in the OLR which might

e due to the higher substrate availability at higher loadingates. While operating at OLR1, the reactor registered a maxi-um COD removal efficiency (74.91%) accounting for an SDR of

.64 kg COD/m3-day under stable conditions. At OLR2, the sys-em documented a maximum COD removal efficiency of 71.61%ith SDR of 1.21 kg COD/m3-day. System showed a COD removal

fficiency of 63.39% and 57.36% at OLR3 and OLR4, respectivelyccounting for SDR of 2.14 kg COD/m3-day and 3.75 kg COD/m3-ay. Maximum SDR of 3.99 kg COD/m3-day was observed withLR5 operation in spite of lower COD removal efficiency

40.56%).

.5. Hydrogen yield with respect to biomolecules removal

Specific hydrogen yield (SHY) found to vary with both sub-trate removal and biomolecules degradation with function ofrganic load. SHY was found to decrease with the increase inhe organic load in all the OLR studied [OLR1, 16.48 mol/kgODR (362 ml/g CODR); OLR2, 14.69 mol/kg CODR (323 ml/g CODR);LR4, 5.49 mol/kg CODR (120 ml/g CODR); OLR5, 4.56 mol/kg CODR

100 ml/g CODR)] except OLR3 where higher SHY (8.39 mol/kgODR; 185 ml/g CODR) was registered. OLR3 operation registeredaximum SHY at 24 h, while all other conditions documented

igher yields at 48 h (Fig. 4). Kim et al. (2008) reported H2 yieldf 128 ml/g CODR with Clostridium KCTC 1875 as biocatalyst andood waste as substrate. The SHY observed in this study with

ixed culture as biocatalyst is comparatively higher especiallyt lower loading conditions. Decrement of SHY at higher loadingondition might be attributed to complexicity of wastewater dueo increases in organic load. On the contrary, SHY with respecto carbohydrates showed higher SHY at 24 h in all the casesxcept OLR2 and OLR5 where the SHY was higher at 48 h [OLR1,8.31 mol/kg CHOR; OLR2, 35.81 mol/kg CHOR; OLR3, 33.62 mol/kgHOR; OLR4, 25.40 mol/kg CHOR and OLR5, 13.94 mol/kg CHOR]Fig. 4b). Maximum SHY was observed between 24 and 48 h of cycle

peration.

Food waste mainly comprises of total carbohydrate contentnd its conversion efficiency to H2 will be lower due to the non-ydrolytic behavior. Generally, carbohydrates in the food waste are

ig. 3. Performance of bioreactor with respect to substrate degradation vs CODemoval efficiency.

Fig. 4. Variations in SHY observed at different OLR with respect to the removal of:(a) COD; (b) total carbohydrates; (c) hexoses; (d) pentoses; (e) proteins.

polysaccharides comprising starch, glycogen, inulin and cellulosealong with hexoses and pentoses. Starch is present in plants andvegetables and it is converted into simple products by the actionof hydrolytic enzymes during microbial metabolism. Contrast tostarch, cellulose is a polymer of �-d-glucose, insoluble in natureand the major structural component of plants and few vegetables.Cellulose can be hydrolyzed to its constituent glucose units throughmicrobial metabolism. Inulin is a polysaccharide present in someplants; on hydrolysis it produces the simple sugars like fructose.Hydrolysis of polysaccharides to simple sugars is a complex processand is not feasible in all bacteria. Moreover, major constituents ofcarbohydrates are in the cooked form which makes the hydroly-sis simple. Higher SHY based on hexose was observed at 48 h forOLR2 (103.99 mol/kg HexoseR), OLR3 (71.64 mol/kg HexoseR) andOLR5 (26.97 mol/kg HexoseR) and at 24 h for OLR1 (139.24 mol/kgHexoseR) and OLR4 (59.73 mol/kg HexoseR) (Fig. 4c). Degradation ofhexoses to simpler forms also generates energy which may requirefor cell growth and maintenance. Even though hexoses are simpler,SHY was observed to drop at higher OLR which might be due to theload shock. After hexoses, pentoses showed higher SHY due to theirrelatively simple structure which can be easily converted into otherintermediates through the pentose phosphate anabolic pathway(PPP) found in most organisms. The oxidative branch of this path-way consists of two enzymes, glucose 6-phosphate dehydrogenaseand 6-phosphogluconate dehydrogenase, that together produce2 moles of NADPH and 2 moles of ribose 5-phosphate from 1 moleglucose 6-phosphate. The non-oxidative branch carries out one andtwo carbon transfers that convert pentoses to fructose 6-phosphateand glyceraldehyde 3-phosphate. Since fructose 6-phosphate isisomerized to glucose 6-phosphate by glucose 6-phosphate iso-merase, these sugars can be recycled back into the pathway togenerate reducing equivalents which are the basis for H2 produc-tion. Higher SHY was observed with pentose at 24 h in OLR2 andOLR4, while it was at 48 h in the case of OLR1, OLR3 and OLR5.[OLR1, 108.26 mol/kg PentoseR; OLR2, 104.65 mol/kg PentoseR;OLR3, 69.13 mol/kg PentoseR; OLR4, 58.56 mol/kg PentoseR; OLR5,30.15 mol/kg PentoseR] (Fig. 4d). Pentoses also showed similarpattern of decreasing SHY with increasing OLR. Compared to the

individual carbohydrates, proteins showed lower SHY as the degra-dation of proteins takes longer time. But when compared with totalcarbohydrates, proteins showed higher SHY and this might be dueto the presence of insoluble polysaccharides in the total carbohy-

3 Biotechnology 155 (2011) 387– 395

dbsew(O(

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di5tHcbtrtomtrbaatscfo

0

100 0

200 0

300 0

400 0

0 2000 40 00 600 0 80 00 1000 0Initial carbohydrate concentration (mg/l)

Car

bohy

drat

e re

mov

al (m

g/l)

y = 0.3211x + 785.61 R2 = 0.879 9

y = 0.46 77x + 365. 74R = 0.9994

0

1000

2000

3000

4000

0 20 00 400 0 6000 8000Initial carb ohydrate concen trat ion (mg/l)

Car

bohy

drat

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mov

al (m

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y = 0. 6812x + 141.91R = 0.9739

0

500

1000

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0 500 1000 1500 20 00 250 0 30 00 350 0 4000Initial protein concentra tion (mg/l)

Prot

ein

rem

oval

(mg/

l)

y = 0.8009x + 17.159R = 0.9988

0

500

1000

1500

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0 500 1000 150 0 2000 2500 3000Initial protein conce ntration (mg/ l)

Prot

ein

rem

oval

(mg/

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c

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a

Fig. 5. Correlation between: (a) initial carbohydrate concentration and carbohy-

92 M.V. Reddy et al. / Journal of

rates composition which are not easily biodegradable as proteinsy the biocatalyst. Typically, SHY with respect to protein removalhowed higher value at later phase indicating their utilization afterxhaustion of simple sources like hexoses and pentoses. Higher SHYas registered at 48 h with OLR1 (109.71 mol/kg ProteinR), OLR2

75.25 mol/kg ProteinR) and OLR3 (45.53 mol/kg ProteinR), 72 h atLR4 (30.59 mol/kg ProteinR) and OLR5 (10.73 mol/kg ProteinR)

Fig. 4e).The correlation between carbohydrate and protein loads, their

emoval for all five OLR and four lower OLR was evaluated todentify the contribution of carbohydrates and proteins removal in

2 production. Carbohydrates showed lower R2 value at five OLR0.8799) and higher R2 value at four OLR (0.994), indicating thathe lower OLR showed higher removal and involved in H2 produc-ion while higher OLR showed both lower removal efficiency andesser H2 production (Fig. 5). Degradation of carbohydrates showedigher H2 production compared to proteins. Where the R2 value ofroteins was almost similar for both four lower OLR (0.9988) andll five OLR (0.9739). Even though R2 value was same for highernd lower OLR, H2 production was low in case of higher OLR. Thisndicates that the degraded proteins are not completely involved in

2 production and mainly involved in the maintenance of systemuffering capacity.

.6. Total solids removal

Generally canteen waste consists of both soluble and insolu-le solids in higher concentrations. In the study, TS concentrationaried between 1512 mg/l and 11,458 mg/l from OLR1 to OLR5Fig. 2e). Solids removal efficiency was relatively higher, but wasependent on the inlet solid/substrate loading conditions applied.t OLR1, removal efficiency of 65.54% was observed accounting for

total removal of 991 mg/l. With increase in the OLR increase inolids removal was noticed even though the removal percentageecreased [OLR5, 4569 mg/l (39.87%); OLR4, 4087 mg/l (47.67%);LR3, 2893 mg/l (50.87%); OLR2, 1608 mg/l (52.62%)].

.7. Bioprocess analysis

pH, VFA and total alkalinity profiles were monitored along withOD for the evaluation of bioprocess during the experimental run.

.7.1. Redox conditionDrop in the pH profiles during bioreactor operation showed a

istinct trend towards acidification (Fig. 6a). OLR1 showed decreas-ng trend in pH from 6 to 4.5 (at 48 h) followed by inversion to pH.2 (at 72 h). All the other OLR showed decreasing trend from 0 ho 72 h (OLR2, 6–4.6; OLR3, 6–4.2; OLR4, 6–4.3 and OLR5, 6–4.2).owever, variations in pH were observed in a narrow range indi-ating the stability of the system. Optimum pH for methanogenicacteria is between 6.0 and 7.5, while acidogenic bacteria func-ions well below pH 6 (Venkata Mohan, 2009). Acidogens growelatively faster and are less sensitive to pH variation than ace-ogens/methanogens. This usually results in the accumulation ofrganic acids and lowering of pH, lead to the suppression ofethanogenic activities. Drop in pH from 6 to 4.2 was considered

o be an ideal pH range for the functioning of the acidogenic bacte-ia and at the same time for inhibiting the activity of methanogenicacteria. The necessity to maintain reactor operating environmentt pH 6.0 was to facilitate the inhibition of methanogenic bacteriand at the same time promoting the prevalence of acidogenic bac-eria by the stabilized acid production and utilization within the

ystem. The observed pH drop during the reactor operation can beonsidered to be favorable for effective H2 yield and for the effectiveunctioning of the acidogenic bacteria coupled with the inhibitionf methanogenic bacteria.

drate removal for five OLR; (b) initial carbohydrate concentration and carbohydrateremoval for four OLR; (c) initial protein concentration and protein removal for fiveOLR; (d) initial protein concentration and protein removal for four OLR.

3.7.2. Volatile fatty acidsFermentative H2 production is generally accompanied by acid

and solvent production. Generation of the acidic intermediatescauses changes in the metabolic pathway of the microorganismsinvolved and their estimation provides a better understanding ofthe system which can be used to improve H2 production. pH dropin the system especially in anaerobic microenvironment was con-

sidered as an index of VFA with the existing buffering capacity(alkalinity) of the system (Fig. 6b). It is interesting to note thatthe VFA production varied consistently with the substrate concen-tration. VFA profile showed higher value at increasing operation

M.V. Reddy et al. / Journal of Biotech

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inity

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c

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ig. 6. Change in; (a) pH; (b) VFA; (c) alkalinity with the function of different organicoading rates.

ime with increasing OLR. OLR1 showed higher VFA production at6 h, while OLR2 and OLR3 showed at 48 h and was extended to2 h in the case of OLR4 and OLR5. During OLR1 operation, VFA0 h; 185 mg/l) production increased to 925 mg/l (36 h), followed

ig. 7. Variations in SHY observed at different OLR with respect to the removal of biomoH by surface plots.

nology 155 (2011) 387– 395 393

by decrement at 72 h (319 mg/l). Similarly, higher VFA generationwas noticed in the case of OLR2 (0 h; 512 mg/l) and OLR3 (0 h;2029 mg/l) which showed an increment up to 48 h (2220 mg/l and3820 mg/l) followed by a drop (72 h; 1421 mg/l and 2628 mg/l)respectively. In the case of OLR4 (0 h, 1196 mg/l) and OLR5 (0 h,1324 mg/l) there is a steady increment in VFA concentration up to72 h (3648 mg/l and 4210 mg/l) respectively. At lower OLR, com-paratively low yield of VFA production was observed, which mightbe attributed to the low substrate availability. Higher VFA con-centration was observed at OLR5 where H2 production droppeddrastically indicating the negative impact of acid metabolites onthe H2 metabolism. H2 yields also decreased with increasing OLRin association with increasing VFA concentration resulting in theacidification of redox microenvironment. This phenomenon shiftsthe metabolic path from acidogenesis to the other pathways.

3.7.3. Buffering capacityAlkalinity indicates the buffering capacity of an anaerobic

bioreactor in association with redox microenvironment and VFAconcentration (Kim et al., 2006; Kraemer and Bagley, 2007; Idaniaand Hector, 2009). Buffering capacity of a system maintains stableionic strength against changes in the acidic and basic constituents.Moreover, alkalinity plays a vital role in restricting the organic acidaccumulation leading to a balanced pH level within the reactor to

enable H2 production coupled with substrate removal during thereaction. pH and VFA profiles corroborated well with each other.Alkalinity concentration showed a gradual decrement from 0 to72 h in all the cases except with OLR1 operation (Fig. 6c). Maximum

lecules (total carbohydrates, hexoses, pentoses, proteins) and VFA generation with

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lkalinity was observed at the beginning of the reactor operation atLR1 (240 mg/l) followed by a gradual drop up to 48 h (200 mg/l)hich later increased till 72 h (290 mg/l). In the remaining OLR

perations also the same pattern of decreasing concentrationrom 0 to 72 h (OLR2, 275–220 mg/l; OLR3, 375–290 mg/l; OLR4,50–480 mg/l; and OLR5, 820–670 mg/l) was noticed. Due to acidccumulation a gradual reduction in the system buffering capacitytotal alkalinity) was observed, which resulted in a concomitantecline in the system pH. In spite of major variations in alka-

inity during different OLR studied, alkalinity was found to havettained a better balance among the acid producers and acid utiliz-rs, leading to higher COD reduction and lower VFA accumulationuring the stabilization phase. Proteins also played a crucial role

n maintaining both system redox condition and buffering capacityy producing bicarbonate ions. Due to the presence of this ions theeactor showed very well buffering capacity and the values wereaintained up to end of the experiment with less decrement. The

xperimental conditions thus prevented complete acidification ofhe reactor and lead to efficient H2 production.

.8. Surface methodology

Surface plots were drawn using Minitab 16 software. SHYas taken as the constant Z-variable coefficient and carbohydrate

emoval (CHOr), CODr, hexose removal (HXr), pentose removalPNTr), protein removal (PRTNr), VFA and pH variations observedt different organic loadings were taken as X and Y variable coef-cients (Fig. 7). From the surface plots, the removal pattern ofiomolecules with respect to VFA generation and pH variationsn SHY at different OLR was studied. A similar trend in the SHYas observed when surface plots were plotted in the combinations

f SHY-CODr-CHOr, SHY-PRTNr-CODr and SHY-HXr-PNTr i.e., withncrease in the OLR, SHY was found to be decreased and the concen-ration of biomolecules removal varied depending upon the OLR.

aximum SHY was observed in the case of OLR1 among all the OLRtudied. A different trend in the surface plots was noticed whenHY-pH-VFA was taken. Maximum VFA generation was observed inhe case of OLR3 followed by OLR5 and OLR4. Minimum VFA gener-tion was observed in the case of OLR1 followed by OLR2. Variationn the VFA generation was mainly found to vary depending on theLR and this might be the reason for minimum generation of VFA

n the case of OLR1. But the trend in SHY followed the same i.e.,ith the increase in the OLR, SHY was found to decreased. pH was

round 4.2–4.5 when the VFA production was maximum in all theLR. The plots depict that how much removal of the substrate com-ounds and formation of VFA and pH conditions favors SHY. As theoncentrations of VFA are increasing the pH gets decreased favor-ng acidogenic fermentation for H2 production. Overall from theurface plots, it is clearly visualized how the trend was followedt different OLR for SHY. Depending upon the OLR given, the influ-ncing parameters removal was varied resulting in the SHY at thatarticular mode of operation (Fig. 7).

. Conclusions

This study demonstrated the functional role of carbohydratesnd proteins present in the canteen based food waste on thecidogenic H2 production process. Total carbohydrates, hexoses,entoses, proteins and total solids showed good reduction during2 production.

Carbohydrates especially hexoses involved in the H2 produc-

ion, while the proteins played a major role in maintaining theystem buffering capacity. H2 generation was found to depend onhe substrate load under selected reactor operating conditions viz.,cidophilic pH, batch mode operation and anaerobic mixed cul-

nology 155 (2011) 387– 395

ture as biocatalyst. Decrement in H2 production was observed withincreasing organic load. However, operation at higher loading ratesshowed improved treatment efficiency due to good biodegradablenature of the canteen waste.

Acknowledgements

The authors wish to thank the Director IICT for his supportand encouragement in carrying out this work. MVR and KC grate-fully acknowledges the Council of Scientific and Industrial Research(CSIR), New Delhi, for providing research fellowship.

Funding: The work was supported by Ministry of New and Renew-able Energy (MNRE), Government of India as national mission modeproject on hydrogen production through biological routes (No.103/131/2008-NT).

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