Bioresource Technology 102 (2011) 2773–2780

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Bioresource Technology

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Assessment of optimum dilution ratio for biohydrogen production by anaerobicco-digestion of press mud with sewage and water

B. Radjaram a,⇑, R. Saravanane b,1

a Department of Civil Engineering, Pondicherry Engineering College, Puducherry 605 014, Indiab Environmental Engineering Laboratory, Department of Civil Engineering, Pondicherry Engineering College, Puducherry 605 014, India

a r t i c l e i n f o

Article history:Received 21 March 2010Received in revised form 16 November 2010Accepted 17 November 2010Available online 26 November 2010

Keywords:Press mudSewageUASBBiohydrogenCo-digestion

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.11.075

⇑ Corresponding author. Tel.: +91 413 2643007,fax: +91 413 2643008.

E-mail addresses: radjaram@rediffmail.com (Bgmail.com (R. Saravanane).

1 Tel.: +91 413 2655281x210, mobile: +91 93451 56

a b s t r a c t

Anaerobic co-digestion of press mud with water or sewage at ratios of 1:7.5, 1:10 and 1:12.5 were per-formed in continuously fed UASB reactors for hydrogen production. At a constant hydraulic retentiontime of 30 h, the specific hydrogen production rate was 187 mL/g volatile solids (VS) reduced during max-imum biohydrogen production of 7960 mL/day at a 1:10 ratio of press mud to sewage. Chemical oxygendemand (COD) and VS reductions of 61% and 59% were noted on peak biohydrogen yield. A pH range of5–6 was suitable at ambient temperature for entire process; a lower pH was inhibitory. Co-digestion ofacidic press mud with sewage controlled pH for fermentation. Hence press mud can be exploited for bio-hydrogen production.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Hydrogen is a promising alternative to carbon-based fuels be-cause it is clean, renewable and has a high energy yield of122 kJ/g (Chang and Lin, 2004). At present, hydrogen is producedmainly from fossil fuels, biomass and water using chemical or bio-logical processes. One such biological process is anaerobic hydro-gen fermentation which can utilize highly concentrated organicwastewater and biomass, such as municipal solid wastes and sew-age sludge as raw material (Das and Veziroglu, 2001). Press mud isanother potential waste source for hydrogen production. About 4%of crushed sugar cane is converted to press mud, and annuallyabout 4.2 million tons of press mud is available from the sugarindustry in India (Saravanane and Radjaram, 1998). It is an acidic,thus mildly corrosive, compressed fibrous waste that contains5–15% sugar (Partha and Sivasubramanium, 2006) and 84% is bio-degradable. Earlier studies on press mud examined the feasibilityof methane production by methanogenesis at pH of 8 (Saravananeand Radjaram, 1998). In contrast, hydrogen production is favoredat an acid pH (Gomez et al., 2006; Mohanakrishna et al., 2010).

As a semi-solid material, press mud cannot be fermented unlesswater or sewage is added. Fermentation can be carried out in an

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mobile: +91 94437 48471;

. Radjaram), saravananae@

037; fax: +91 413 2655101.

upflow anaerobic sludge blanket (UASB) process since this anaero-bic treatment system has a high treatment efficiency and a shorthydraulic retention time (HRT) (Chang and Lin, 2004). Fermenta-tion in a continuous stirred tank reactor does not seem practicalsince this type of reactor was unable to maintain high level of fer-mentative biomass for hydrogen production and its specific hydro-gen production rate was low (Yu et al., 2002). Dilution is a keyparameter in anaerobic fermentation as low dilution leads to prod-uct inhibition and high dilution leads to wash out of biomass inreactor (Han and Shin, 2004). In the present study water and sew-age were used to dilute press mud to allow efficient fermentation.

2. Methods

2.1. Seed

Seed sludge was collected from a municipal wastewatertreatment plant maintained by the Public Works Department,Pondicherry, India. It was sieved through a wire mesh of Ø0.5 mm to remove solid materials that may block the flow in thepump. Cow dung was mixed with water at a 1:2 ratio and digestedunder anaerobic conditions for 30 days at a pH between 5 and 6 byadding HCl/NaOH as needed. The digest was filtered through wiremesh (Ø 0.5 mm) to remove fibrous materials, mixed with seedsludge at 4:1 ratio, and heated to 70 �C for 1 h to inhibit themethanogens, and used as seed. The characteristics of the seed(fermented cow dung diluted with sludge) are given in Table 1.

Table 1Characteristics of seed and feeds of R1 and R2 for dilutions of 1:7.5, 1:10 and 1:12.5.

Substrate pH TS TDS TSS TVS TVDS TVSS Total COD Alkalinity VFA

Seed 6.76 10.9 2.05 8.85 6.8 1 5.8 10.40 19.03 1.82Reactor R11:7.5 6.46 7.7 2.25 5.45 4.65 1 3.65 12.48 12.68 1.501:10 5.01 7.7 2.45 5.25 5.5 2.4 3.1 17.88 15.85 5.221:12.5 5.65 15.4 2.2 13.2 4.2 0.8 3.4 29.95 6.34 1.68

Reactor R21:7.5 5.2 9.9 7.6 2.3 4.25 2.27 1.98 10.56 27.36 2.041:10 5.18 7.2 1.5 5.7 3.9 0.5 3.4 32.03 47.55 0.341:12.5 6.12 6.6 5.62 0.98 3.2 2.48 0.72 8.32 19.03 5.52

All units are in g/L except pH.

2774 B. Radjaram, R. Saravanane / Bioresource Technology 102 (2011) 2773–2780

2.2. Feed substrate

The characteristics of press mud and sewage are given in Table 2.In preliminary experiments it was determined that a minimumdilution of 1:7.5 of press mud and water was necessary to obtaina hydrogen fermentation substrate that can be filtered and ulti-mately used in a continuous flow system. Therefore, feeds for reac-tor R1 were prepared for three series of experiments by diluting4 kg press mud with 30, 40 and 50 L of water to obtain dilutionratios of 1:7.5, 1:10, and 1:12.5, respectively. Feeds for reactor R2were prepared at the same dilution ratios by adding sewage insteadof water. The press mud/sewage and press mud/water mixtureswere allowed to soak for 4 h, filtered through wire mesh(Ø 0.5 mm) to remove fibers and the filtrate was fed into the reac-tors. The characteristics of the feed substrate are given in Table 1.

2.3. Bioreactor system

Two UASB reactors of volume 20 L each were used in this study.The cylindrical plexiglas reactors had an internal diameter 10 cmand a height of 190 cm (H/D > 10) with a gas liquid separator atthe top (diameter, 20 cm; height, 20 cm) with an effluent port at15 cm from the bottom of the gas liquid separator. Seven samplingports were evenly fixed over the entire height of the column at25-cm interval. The gas liquid separator housed an inverted funnelconnected to a wet gas meter through a flexible tube for measuringthe gas flow rate. The reactor had provisions for feeding from thebottom and removal of effluent from the top. The reactor receivedfeed from a feed tank through a peristaltic pump (Ravel RH P 100 L)as shown in the schematic diagram (Fig. 1). The feed substrate inthe feed tank was stirred at every 20 min for 3 min with a mechan-ical stirrer fitted in the tank to prevent settlement of solids.

Table 2Characteristics of press mud and sewage.

Parametersa Press mud Sewage

pH 4.5–5 7.2COD (%) 117.6 13.6C/N ratio 24.04 17.5Total solids (%) 29 7.4Moisture content (%) 71 –Total volatile solid (%) 84 5.8Organic carbon (%) 48.80 6.1Nitrogen (%) 2.05 1.96Phosphorous (%) 0.65 –Potassium (%) 0.28 –Sodium (%) 0.18 –Calcium (%) 2.7 –Sulphate (%) 1.07 –Sugar (%) 3 –Wax (%) 1 –

a Average values.

2.4. Operation

Reactors R1 and R2 were operated with press mud diluted withwater and sewage, respectively. HRT was fixed at 30 h since a high-er HRT does not lift the particulate substrate in the reactor column.The experiments were started in R1 and R2 with a 1:7.5 dilutionand performance was observed till steady state was reached inboth reactors. The operation was continued at steady state for 30additional days. Then, the dilution was changed to 1:10 and theperformance was monitored for 30 days. Finally the dilution waschanged to 1:12.5 for another 30–34 days. The pH of the reactorswas between 5 and 6, by adding HCl/NaOH as needed with the feedand the reactors were operated at ambient temperatures(30–38 �C) without heating or cooling.

2.5. Analytical methods

Temperature, pH and biohydrogen production were measureddaily with a thermometer, pH probe and wet gas meter, whilethe gas composition was analyzed using a gas chromatograph(Nucon GC). The total volatile solids (VS), chemical oxygen demand(COD), volatile fatty acid (VFA), alkalinity, etc. were estimated oncea week by standard methods (APHA, 1995).

3. Results and discussion

3.1. Start-up of UASB reactor

During the start-up period, the UASB system was fed continu-ously with diluted substrate at a concentration starting from2500 to 10,000 mg COD/L to reach an organic loading rate (OLR)of 8–20 g/L/day. The pH in the reactor decreased from 6.5 to 5.5during the start-up period, while hydrogen production (Fig. 2b)and COD removal efficiency (Fig. 2c) gradually increased. Biohy-drogen production started on day 29 in R1, with an uneven outputfor 76 days. After 76 days, both biohydrogen production and CODremoval efficiency stabilized in the system, reaching approxi-mately 1600 mL/day and 70%, respectively (Fig. 2b and c). Hydro-gen production started on day 34 in R2 with an unsteady outputup to day 60. After 60 days both hydrogen yield and COD removalefficiency stabilized. These results revealed that anaerobic acti-vated sludge of reactors R2 and R1 possessed good acid-toleranceand stable hydrogen production ability, which indicated goodacclimatization.

3.2. Continuous hydrogen production

R1 reached steady-state conditions, defined as consistenthydrogen production with a variation of less than 10%, after90 days (Fig. 2b). The hydrogen production was 1900 mL/day witha 1:7.5 diluted feed. When, the dilution was changed to 1:10 on

Ø100

Feed tank

200 L

Timer

Drain

Gas liquid separator

Sampling ports

Effluent port

Gas meter

Peristaltic pump

1900

Ø 200

150200

250

Feed

All dim in mm

Fig. 1. Schematic arrangement of UASB reactor for biohydrogen production.

B. Radjaram, R. Saravanane / Bioresource Technology 102 (2011) 2773–2780 2775

day 120, production gradually reached 5000–6000 mL/day by day136. After 144 days it reached 7210 mL/day and finally attained amaximum of 7820 mL/day during days 145–150 (Fig. 2b). Whenthe dilution was changed to 1:12.5 on day 150, gas production de-clined and did not stabilize (Fig. 2b). Maximum hydrogen produc-tion was 4.887 L/kg press mud at 1:10 dilution in R1. Reactor R2took 60 days to reach steady-state conditions. At a 1:7.5 pressmud:sewage ratio, maximum hydrogen production was 1990 mL/day on day 89. When the dilution was changed to 1:10 on day90, the gas yield dropped below 1000 mL/day for 1 week time,but increased thereafter (Fig. 4b). The highest hydrogen productionof 7960 mL/day occurred on day 115. At a dilution of 1:12.5, max-imum hydrogen production was 3880 mL/day (Fig. 4b). Maximumhydrogen of 4.990 L/kg press mud added was evaluated at a 1:10dilution in R2. These yields are lower than those obtained withwheat feed and sweet sorghum fermentations which were 56 L/kg feed at HRT of 15 h (Hawkes et al., 2008) and 10.41 L/kg feed(Georgia et al., 2008), respectively; however, the use of such sub-strates for hydrogen production is questionable since they are alsoused as animal feed. The yields were higher than those achievedwith Rhodobacter sphaeroides RV which reached only 1.4–1.6 L/Lreactor volume/day (Fascetti and Todini, 1995) and 1.3 mL/mL por-ous glass media/h (Tsygankov et al., 1994) and required input oflight. Earlier studies had demonstrated that sewage sludge diges-tion can produce hydrogen at the rate of 3.75 mL/min (Nicolauet al., 2008) and that acclimatized anaerobic activated sludge hada hydrogen producing ability as high as 10.4 m3 H2/m3 reactor/day in a continuous reactor with an available volume of 9.6 L(Ren et al., 1995. The co-digestion experiments with press mudand sewage demonstrated that sewage was able to support

hydrogen production since it yields 4.99 L/kg press mud feed, i.e.0.5 m3/m3 reactor volume/day.

H2 % gradually increased with experiment days and reached amaximum of 50–55% during consistency periods of study in reac-tors 1 and 2 (Figs. 2 and 4b).

3.3. Assessment of COD loading rate, % reduction and specific hydrogenproduction rate

COD removal fluctuated in the initial start-up stage and laterstabilized after 80 days of operation in R1 (Fig. 2c). COD reduc-tion was 60–77%, 70% and 86% at 1:7.5, 1:10, and 1:12.5 dilu-tions, respectively (Fig. 2c). The high COD reduction waspossible due to the large column height (H/D > 10) which pro-vided a long mixing path during substrate flow from inlet tooutlet. Filling the column to about one third its heights withbiomass allowed maintenance of adequate numbers of hydro-gen-producing bacteria in the reactor. Specific biohydrogen pro-duction rates (SBPR) were 10.98, 40.6 mL, and 2.58 mL/g CODreduced/day at maximum hydrogen production at 1:7.5, 1:10,and 1:12.5 dilution, respectively (Fig. 3a). In R2, COD loadingrates varied between 6 and 25 g/L/day (Fig. 4c) and COD reduc-tions were 36–41%, 55–61%, and 40–41% at the 1:7.5, 1:10, and1:12.5 dilutions, respectively (Fig. 4c). Anaerobic fermentation offood waste in leaching bed reactor gave biohydrogen yield of21.2–41.5 mL/g COD at an HRT of 25 h (Kim et al., 2004). Biohy-drogen yield was 26.13 mol/kg COD reduced in molasses fer-mentation (Ren et al., 2006) and 1.8–2.3 mM/g COD fed incheese processing waste water (Yang et al., 2007) whereas inour study 41 mL/g COD reduced was achieved from a waste

Fig. 2. (a) Duration of experiments, (b) biohydrogen yield and % H2, (c) COD loading rate and % reduction, (d) VS loading rate and % reduction of reactor R1 digesting pressmud and water.

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substrate by co-digestion. SBPR in R2 was 20–37, 35–41, and39–51 mL/g COD reduced/day at 1:7.5, 1:10, and 1:12.5 dilu-tions, respectively (Fig. 5a). With a decrease in the dilution ra-tio, substrate becomes dense and product inhibition occurs

due to excess VFA accumulation in the reactor, but higher dilu-tion led to wash out of bacteria and decreased biohydrogen pro-duction. Han and Shin (2004) observed a similar outcome intheir study.

Fig. 3. Profiles of various operating parameters – (a) SPBR, (b) VSS/TSS and VFA/alkalinity ratios, (c) VFA and (d) alkalinity – monitored during steady state and three series ofexperiments in reactor R1 digesting press mud and water.

B. Radjaram, R. Saravanane / Bioresource Technology 102 (2011) 2773–2780 2777

3.4. Appraisal of Volatile solid loading rate, % reduction and SBPR

Volatile solid (VS) loading rates reached a maximum of 13 g/L/day in R1 (Fig. 2d). VS reduction was 55%, 90%, and 69% at 1:7.5,

1:10, and 1:12.5 dilutions, respectively (Fig. 2d). Hydrogen yieldgradually increased after 75 days and was consistently around1600 ± 150 mL/day at a 1:7.5 dilution with a SBPR of 12.77 mL/gVS reduced/day on peak output. Though the SPBR was as high as

Fig. 4. (a) Duration of experiments, (b) biohydrogen yield and % H2, (c) COD loading rate and % reduction, (d) VS loading rate and % reduction of reactor R2 digesting pressmud and sewage.

2778 B. Radjaram, R. Saravanane / Bioresource Technology 102 (2011) 2773–2780

32.81 mL/g VS reduced/day, it was not constant. SBPR was 62.66and 17.92 mL/g VS reduced/day at a 1:10 and 1:12.5 dilution,respectively (Fig. 3a). The substrate in the feed tank underwentslow hydrolysis which resulted in VS accumulation during theinitial stages up to 30 days (Fig. 2d). In R2, VS loading rate variedbetween 3 and 16 g/L/day (Fig. 4d). VS reduction fluctuated in

the start-up stage and stabilized under steady state condition. VSreduction was between 53–63%, 57–59%, and 40–57% at the1:7.5, 1:10, and 1:12.5 dilutions, respectively (Fig. 4d). SBPR washighest at the 1:10 dilution as187 mL/g VS were reduced per day(Fig. 5a). Co-digestion of municipal solid waste and slaughterhouse waste gave H2 yield of 52.5–71.3 mL/g VS reduced (Gomez

Fig. 5. Profiles of various operating parameters – (a) SPBR, (b) VSS/TSS and VFA/alkalinity ratios (c) VFA and (d) alkalinity – monitored during steady state and three series ofexperiments in reactor R2 digesting press mud and sewage.

B. Radjaram, R. Saravanane / Bioresource Technology 102 (2011) 2773–2780 2779

et al., 2006) and 9.33 L H2/g VSS/day was observed in rice winerywaste water (Yu et al., 2002), whereas in our co-digestion H2 yieldwas 187 mL/g/VS reduced. As the reactor had a height/diame-ter > 10, 1.9 times more than the normal UASB reactor (Castelloet al., 2009) the biomass concentration increased from the initial

level of 150 to a maximum of 1200 mm. The digested substrateof low density floats and reaches the upper portion of reactor asevident by separation and collapsing of biomass layer in reactorwhich will be removed at regular period. Haandel van and Lettinga(1994) noted similar observation in their study.

2780 B. Radjaram, R. Saravanane / Bioresource Technology 102 (2011) 2773–2780

3.5. Observation of VFA, alkalinity and pH

The VFA concentration in the effluent of R1 was high during thestart-up stage and declined gradually (Fig. 3c) indicating acid tohydrogen conversion after 30 days. The later increase was likelydue to the accumulation of sludge in the reactor by the digestedsubstrate mass. The sludge was removed daily at a rate of 2 L atthe sampling port and this process reduced VFA accumulation tobelow 15 g/L. Before the production increased above 1000 mL/day, a separation or parting of biomass layer in the reactor oc-curred at 300 mm from the inlet port. Subsequently the biomasslevel was separated by 150 mm gap of liquid. This separation oc-curred once in every 3 days showing an effective anaerobic reac-tion by upward movement of digested substrate. The separationlayer lasted for 4–12 h, as the gas rose to the surface and particlessettled. The VFA level was constant at a 1:10 dilution with9516 mg/L (Fig. 3c). Sporadically discharging sludge is a feasiblemeans of dealing with sludge production from a biological system.In order to appraise the performance of a UASB reactor, it is imper-ative to know whether the reactor is operated at its utmost sludgeholdup. Hence, the reactor could be operated by periodically dis-charging excess sludge, before sludge attains a maximum level(Haandel van and Lettinga, 1994). VFA production followed an un-steady profile during start-up in R2, but reached consistency dur-ing steady state gas yield (Fig. 5c). The VFA of the effluent was19,202, 180, and 240 mg/L at 1:7.5, 1:10 and 1:12.5 dilutions,respectively (Fig. 5c).

Alkalinity was lower at the 1:10 dilution in R1 (9516 mg/L) thanat the other dilutions and gave maximum biohydrogen yield(Fig. 3d). In R2, alkalinity levels of the feed fluctuated between60 and 41 g/L during the start-up period, rose to 57 g/L at the1:10 dilution and declined to 19 g/L at the 1:12.5 dilutions(Fig. 5d). The hydrogen yield was maximum at elevated alkalinityof feed (57 g/L) and effluent (38 g/L) while VFA levels were low.

Feed pH was maintained between 5 and 6 by acidic press mud,an added advantage for fermentation. Studies on municipal solidwaste and slaughter house waste fermentation found 5–6 as opti-mum pH (Gomez et al., 2006) and food waste with sewage sludgeco-digestion was at a pH of 6 (Kim et al., 2004) indicating particu-late substrates requires a pH range of 5–6. Due to anaerobic fer-mentation, the pH of the effluent was always near 6.

3.6. Assessment of VSS/TSS and VFA/alkalinity ratios

The volatile suspended solids to total suspended solids ratio(VSS/TSS) in the UASB reactor denotes the biomass componentsof the sludge and variation in VSS/TSS indicates change in biomasscomponents. A VSS/TSS ratio of 0.6–0.8 has been reported in UASBreactor treating sewage (Haandel van and Lettinga, 1994) and 0.76for a HRT of 24 h in sucrose treated in a UASB reactor was reportedby Chang and Lin (2004). At a 1:10 dilution, a VSS/TSS ratio of 1.1(Fig. 3b) and 1.45–1.5 (Fig. 5b) gave maximum biohydrogen yieldfor R1 and R2, respectively. At very low VSS/TSS ratios, the hydro-gen yield decreased due to high sludge productivity which affectedthe fermentation process. A low VSS/TSS ratio indicates the organicfraction degradation. VFA/alkalinity ratios of 0.01 for R2 and of 0.03for R1 in 1:10 diluted press mud (Figs. 3 and 5b) provided for max-imum hydrogen yields. The concentration of VFA in the feed and

effluent showed a considerable increase with increasing organicloading rates (Figs. 3 and 5c).

4. Conclusions

Digestion of press mud and sewage at a 1:10 dilution and at a30 h HRT resulted in the production of approximately 2.75 L H2/kgpress mud. As press mud is of no use other than as soil fertilizer, itcould be fully exploited to produce hydrogen. From 4.2 million tonsof press mud available annually in India, it is estimated that11.55 million m3 hydrogen could be produced. Only limited acid/base additions were necessary to maintain a pH of 5–6, resultingin cost savings.

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