Effect of micro-nutrients in anaerobic degradationof sulfate laden organics

S.K. Patidar and Vinod Tare

Abstract: The effect of micro-nutrients, such as Fe, Ni, Zn, Co, and Mo, on anaerobic degradation of sulfate ladenorganics was investigated using bench-scale models of upflow anaerobic sludge blanket (UASB) reactor, anaerobic baf-fled reactor (ABR), and hybrid anaerobic baffled reactor (HABR), operating in varying conditions in ten phases (or-ganic loading of 1.9–5.75 kg COD/(m3·d), sulfate loading of 0.54–1.88 kg SO4

2–/(m3·d), chemical oxygen demand(COD):SO4

2– ratio of 2.0–8.6). In the initial phase, no nutrient limitation was observed with COD removal of morethan 94% in all three systems. Subsequently, increase in sulfate loading resulted in Ni and Co limitation and theirsupplementation restored COD removal in UASB system. However, baffled systems did not recover because of severeinhibition by sulfide. Results indicate that precipitation of nutrients could seriously deteriorate process performance,leading to failure even before sulfide concentration attains toxic level. The limitation of Fe coupled with high sulfateloading (1.88 kg SO4

2–/(m3·d)) resulted in growth of low-density, fragile, hollow, and granular biomass in UASB thatwashed out and caused process instability. Supplementation of Fe with other nutrients stabilized UASB process andalso improved COD removal.

Key words: anaerobic degradation, nutrients, UASB, ABR, HABR, sulfide toxicity, sulfate laden organics.

Résumé : L’effet des micronutriments Fe, Ni, Zn, Co et Mo sur la digestion anaérobie des produits organiques chargésde sulfates a été examiné en utilisant des modèles de réacteurs anaérobie hybride (« upflow anaerobic sludge blanket :UASB »), anaérobie cloisonné (« anaerobic baffled reactor : ABR »), anaérobie hybride cloisonné (« hybrid anaerobicbaffled reactor : HABR »), à l’échelle du laboratoire, fonctionnant sous diverses conditions en 10 étapes (chargementorganique de 1,9 à 5,75 kg DCO/m3·j; chargement en sulfates 0,54 à 1,88 kg SO4

2–/m3·j; rapport DCO:SO42– entre 2,0

et 8,6). Lors de la phase initiale, aucune limitation en nutriment n’a été observée par le retrait de plus de 94 % de laDCO dans les trois systèmes. Ainsi, une augmentation en chargement de sulfates a engendré une limitation en Ni et enCo et leur adjonction a restauré l’élimination de la DCO dans le système UASB. Cependant, les systèmes cloisonnésn’ont pas récupéré en raison de l’inhibition sévère causée par les sulfures. Les résultats indiquent que la précipitationdes nutriments peut sérieusement diminuer le rendement du procédé, menant à une défaillance même avant que laconcentration en sulfures n’atteigne un niveau toxique. La limitation en Fe associée à un fort chargement en sulfates(1,88 kg SO42–/m3·j) a engendré la croissance d’une biomasse granulaire de faible densité, fragile et creuse dans leUASB; cette biomasse a été lavée et a engendré l’instabilité du procédé. L’adjonction de Fe par d’autres nutriments apermis de stabiliser le procédé UASB et a amélioré l’élimination de la DCO.

Mots clés : digestion anaérobie, nutriments, réacteur anaérobie hybride (UASB), réacteur anaérobie cloisonné (ABR),réacteur anaérobie hybride cloisonné (HABR), toxicité des sulfures, produits organiques chargés de sulfates.

[Traduit par la Rédaction] Patidar and Tare 431

Introduction

Anaerobic technology has proven to be a stable processfor a variety of wastes when operated properly with several

advantages over the aerobic and physico-chemical processeslike low sludge production, higher loading potential, low op-erating cost, and methane production. However, sulfide gen-eration along with organic removal during anaerobicdegradation of sulfate laden waste causes many problemsincluding toxicity to microbial consortia. Anaerobic degra-dation of sulfate laden organics involves competitive inter-actions among various groups of bacteria includingfermenters, methane-producing bacteria (MPB), and sulfate-reducing bacteria (SRB). A number of parameters affect thisdelicate ecosystem including availability of nutrients for en-zymatic activity and biomass growth. Advances in microbi-ology have identified trace metals as cofactors orcomponents of prosthetic group of enzymes, and their bio-availability affect functioning of anaerobic digestion sys-tems. Metals present in metalloenzymes or metalloproteinshave different functions: catalytic role, structural role, regu-

Can. J. Civ. Eng. 31: 420–431 (2004) doi: 10.1139/L03-104 © 2004 NRC Canada

420

Received 24 May 2003. Revision accepted 17 November2003. Published on the NRC Research Press Web site athttp://cjce.nrc.ca on 14 May 2004.

S.K. Patidar. Department of Civil Engineering, NationalInstitute of Technology, Kurukshetra 136 119, India.V. Tare.1 Environment Engineering and ManagementProgram, Department of Civil Engineering, Indian Institute ofTechnology, Kanpur 208 016, India.

Written discussion of this article is welcomed and will bereceived by the Editor 31 October 2004.

1Corresponding author (e-mail: vinod@iitk.ac.in).

latory role, and noncatalytic role. Studies have documentedpresence and possible functions of minerals specific to MPB.The functions include enzymatic activity, membrane stabil-ity, nutrient transport, and energy conservation (Takashimaand Speece 1990). Fe is involved in energy metabolism ascytochromes, ferredoxins, and other Fe-S proteins. Co ispresent in corrinoids, which are involved in the activity ofmethyl transferase and carbon monoxide (CO) dehydro-genase. Nickel is component of methyl coenzyme M, factorF420, factor F430, hydrogenase and CO dehydrogenase(DiMarco et al. 1990; Ankel-Fuchs and Thauer 1988). N, P,and S are involved in many coenzymes in addition to theirrole of providing the building blocks required for growth ofMPB. Whitman et al. (1982) reported high Zn levels for allmethanogens tested than the levels of Ni and Co, and it hasbeen found to be present in hydrogenase, CO dehydrogenase,and formate dehydrogenase. Zn is reported to be involved incoenzyme M activation during formation of methyl-coenzyme M (Sauer and Thauer 2000). All methanogens useammonia as nitrogen source. Sulfide is in general a sulfursource for all methanogens, while some species successfullyutilize cysteine or menthionine.

Sulfate reducing bacteria (SRB) also require variousmacro- and micro-nutrients for physiological functions simi-lar to methanogens. Fe is reported to be present in cyto-chrome c3, hydrogenase, APS reductase, bisulfite reductase,formate dehydrogenase, ethanol dehydrogenase, lactate de-hydrogenase, CO dehydrogenase, aldehyde oxidoreductase(Barton 1992; Huxtable 1986). Ni is present in hydrogenase(desulfovibrio desulfuricans), CO dehydrogenase (Ankel-Fuchs and Thauer 1988). Zn and Co are present in COdehydrogenase, ATP sulfurylase (Gavel et al. 1998). Mo ispresent in aldehyde oxido reductase, and aldehyde dehydro-genase.

Nutrient or metal ions bioavailability depends on totalmetal concentration in the substrate, metal precipitation,metal chelation or complexing with both inorganic species(ion pairs) and organic ligands (chelates), including thosesynthesized by microorganisms to assist in metal uptake, andthe kinetics of precipitation and chelation reaction. A unify-ing theory for the microbial availability of metal ions has notbeen reported (Callander and Barford 1983a, 1983b). Thelack of a complete understanding of the various metal trans-

port mechanisms, synergism and antagonism among metalions, identification of metal-specific chelators produced byanaerobic bacteria, and complex role of chelators in uptakeof specific metal ions in a mixed culture makes it difficult toassess requirement and availability of specific nutrient ortheir combinations for optimum growth. A number of stud-ies have been undertaken to assess the effect of nutrientsupplementation on the methanogenesis and sulfidogenesisin batch and continuous systems. Supplementation of Fe, Ni,Co, and Mo has shown positive effects on substrate utiliza-tion, bacterial activity, and process stability (Florencio et al.1994; Gonzalez-Gil et al. 1999; Oleszkiewicz and Romanek1989; Percheron et al. 1997; Shen et al. 1993a, 1993b;Takashima and Speece 1990). Supplementation of Fe has in-dicated positive influence on granulation (Oleszkiewicz andRomanek 1989; Yu et al. 2000). Sulfide precipitation andcomplexation were found to have significant influence onavailability of essential trace metals (Barber and Stuckey2000a, 2000b).

Although these studies have shown positive effects ofmetal supplementation on MPB, SRB, process performance,and process stability, most of them are restricted to batchsystems and continuous flow stirred tank reactors (CSTRs)with methanogenic cultures. Various issues related tonutrient requirements, such as critical nutrients and theircombination(s), effect of sulfide and complexation on bio-availability of metals, effect of system configuration onavailability of nutrients for mixed culture degrading sulfateladen substrate, are yet to be fully elucidated. In view ofthis, the present study was undertaken to assess the effect ofnutrients in the high-rate anaerobic reactors for achievingmaximum organic and (or) sulfate removal. The main focuswas to evolve better understanding of the influence of nutri-ents on metabolization of sulfate laden organics by SRB andMPB to help achieve better process performance.

Materials and methods

Experimental setupFigure 1 shows schematics of the experimental setup. The

setup essentially consists of laboratory scale models ofupflow anaerobic sludge blanket (UASB) reactor having aneffective volume of 3.24 L; anaerobic baffled reactor (ABR)

© 2004 NRC Canada

Patidar and Tare 421

Fig. 1. Experimental setup (GLSS = gas liquid solid separator).

with an effective volume of 5.0 L; hybrid anaerobic baffledreactor (HABR) with an effective volume of 5.0 L, andpolyvinyl chloride (PVC) and rasching rings as carrier me-dia; feed tanks; effluent collection tanks; peristaltic pumps(Janke & Kunkel Gmbh & Co., model IKA-PA-SF5, FRG);gas holders; and wet-type gas flowmeter (INSREF modelIRI 02B, India). The setup was housed in a walk-in con-trolled temperature cabin maintained at 35 ± 1 °C and oper-ated for more than 540 d according to operational conditionssummarized in Table 1.

Process operationThe reactors were seeded with sludge (volatile suspended

solids (VSS) 20.9 g/L, VSS: total suspended solids (TSS) ra-tio of 0.324, and flocculent in nature) collected from aUASB plant treating domestic waste at Kanpur (India). Thesynthetic sulfate-containing wastewater was prepared by dis-solving required quantity of jaggery (a locally available formof sucrose, also known as gur or Indian sugar) in tap water.Sufficient quantity of sodium bicarbonate was added to syn-thetic wastewater (feed) for buffering the systems and main-taining pH above 6.8. Sulfate concentration was maintainedby adding sodium sulfate according to desired COD:SO4

2–

ratio in the influent. No other growth nutrients were addedin the beginning to identify limiting nutrient(s). In subse-quent phases of the study, Fe, Ni, Co, Zn, and Mo were sup-plemented in feed by adding required quantity ofconcentrated stock metal solutions. Usually, a fresh feed wasprepared every 12 h.

All reactors were started simultaneously. However, theperformance of ABR and HABR was very poor with CODremoval of <30% and 37%, respectively. Hence, ABR andHABR were restarted with fresh seed on 83rd day. The timescale of operation and monitoring was reset for the purposeof convenience. The study involved reactor operation in 10phases (A to J) with different influent COD concentrations,sulfate concentrations, COD:SO4

2– ratio, organic loadingrate (OLR), and sulfate loading rate (SULR) with and with-out growth nutrients supplementation in feed. The reactorstart up was accomplished in phase A with sulfate free syn-thetic substrate. Initial hydraulic retention time (HRT) was40 and 30 h for UASB and baffled reactors, respectively.Then, HRT for baffled reactors was reduced to 20 h. Inphase B, reactor operation was started for sulfate containingsubstrate with SO4

2– addition at 0.36 and 0.16 g/L in the be-ginning of the phase for UASB and baffled reactors, respec-tively. The SO4

2– was increased in steps to a finalconcentration of 0.507 g/L for all three reactors. In UASB,HRT was reduced to 20 h and COD concentration was in-creased to increase OLR. In phase C and D, SO4

2– concen-tration was further increased to 0.607 g/L. Nutrients, Ni andCo, were also supplemented in phase D at 0.5 and 0.2 mg/L.In phase E, SO4

2– concentration was further increased to0.707 g/L and Zn supplementation started at 0.5 mg/L. Thesupplementation of Mo at 0.2 mg/L started in phase F withan increase in Co and SO4

2– concentrations to 0.3 mg/L and0.957 g/L, respectively. In phase G, Fe supplementationstarted at 0.25 mg/L with increased SO4

2– concentration of1.457 g/L in feed. Hydraulic retention time (HRT) wasincreased to 30 h in phase H to reduce loading rate for re-covery of process performance. In phase I, Fe and SO4

2–

© 2004 NRC Canada

422 Can. J. Civ. Eng. Vol. 31, 2004

Dur

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concentrations increased to 0.5 mg/L and 2.207 g/L, respec-tively. Supplementation of external nutrients except Fe andCo was withheld in the last phase J of the study to verifybatch assay results in reactor systems.

Chemical analysisThe pH was measured using a digital pH meter (model

CL-54, manufactured by Toshniwal Instruments Manufac-turing Private Ltd., India) according to 4500-H+ B electro-metric method (APHA 1998). Conductivity was measuredusing a digital conductivity meter (model CC 601; manufac-tured by Century Instruments Pvt. Ltd., India) according to2510 B laboratory method (APHA 1998). The COD of pre-treated samples was estimated according to 5220 C closedreflux, titrimetric method (APHA 1998). The samples wereclose refluxed in HACH COD reactor (model 45600; manu-factured by HACH Company Iowa, USA). Samples werepretreated by acidification to pH 2 using orthophosphoricacid and purging with nitrogen for stripping of sulfides forCOD estimation. The concentration of sulfate was deter-mined according to 4500-SO4

2– D gravimetric method withdrying of residue (APHA 1998). Samples were pretreatedfor sulfide determination according to 4500-S2– C samplepretreatment to remove interfering substances or to concen-trate the sulfide (APHA 1998). Dissolved sulfide inpretreated samples was determined according to 4500-S2– Fiodometric method (APHA 1998). Unionized H2S was cal-culated according to procedure given in 4500-S2– H calcula-tion of unionized hydrogen sulfide (APHA 1998); TSS weredetermined according to 2540 D total suspended solids driedat 103–105 °C (APHA 1998). The volatile suspended solidswere determined according to 2540 E fixed and volatile sol-ids ignited at 550 °C (APHA 1998). Direct titration methodgiven by DiLallo and Albertson (1961) was used to measuretotal alkalinity (TA) and volatile fatty acids (VFA) concen-tration. Metals were analyzed using an electrothermal atomicabsorption spectrometer (Varian model Spectra AA20 BQ;GTA-96, Australia).

Methane content of biogasA simple experimental setup, comprising an aspirator bot-

tle to serve as a gas holder, a two-stage CO2 scrubber, and aliquid displacement system, was used for methane content

estimation. A portion of the biogas was transferred to the as-pirator bottle (having acidified water with methyl orange in-dicator) by withdrawing equivalent liquid volume. A knownvolume (100 mL) of biogas was displaced from aspiratorbottle, and it bubbled through scrubbing solution (two-stageCO2 scrubber in series having 32.5% w/v potassium hydrox-ide (KOH) solution and thymol blue as indicator). Thescrubbing solution absorbs CO2 only, whereas methanealong with other trace gases, if any, passes to liquid dis-placement system. The equivalent volume of liquid dis-placed is measured to determine methane content. Thisprocedure was repeated 7–10 times and an average of thelast 3 values was obtained.

H2S content of biogasThe H2S content of biogas was estimated after it was precip-

itated as zinc sulfide using a simple experimental setup thatconsisted of an aspirator bottle to serve as a gas holder and asulfide absorbing stage. A portion of the biogas was transferredto the aspirator bottle (having acidified water with indicatormethyl orange) by withdrawing equivalent liquid volume. Aknown volume (100 mL) of biogas was displaced from an aspi-rator bottle, and it bubbled through zinc acetate solution. Sul-fide was absorbed and precipitated as zinc sulfide in theabsorbing solution. Total sulfide in absorbing solution was esti-mated as per Standard Methods (APHA 1998), and finally hy-drogen sulfide content (% v/v) in biogas was calculated usingtotal sulfide content in absorbing solution and volume of gas.

Scanning electron microscopic analysisSamples were prepared for scanning electron microscopy

(SEM) by fixing with glutaraldehyde, washing with phos-phate buffer, and dehydrating with acetone as per the proce-dure suggested by Glauert (1974). The prepared sampleswere mounted on sample holders (aluminum or brass) forcoating with silver in a sputter coating unit and scanned us-ing a scanning electron microscope (model Jeol JSM840A,Japan) at an accelerating voltage of 5–15 kV.

Electron flowElectron flow designates the ratio of COD scavenged by

MPB and SRB to total COD removed. Electron flow to MPBand SRB was calculated using eqs. [1]–[4] as follows (Isa etal. 1986):

[1] Electron flow distributed to MPB = (CH4(gas-COD) + CH4(aq-COD)/(CH4(gas-COD) + CH4(aq-COD) + ∆SO42–

(COD)

[2] Electron flow distributed to SRB = (∆SO42–

(COD)/(CH4(gas-COD) + CH4(aq-COD) + ∆SO42–

(COD)

[3] COD removal through methanogenesis = CH4(gas-COD) and CH4(aq-COD) = QgMg + QeMe

where Qg is gas flow of CH4 produced (litres per day), Mg is COD of methane gas (= 2.53248 g/L), Qe is effluent flow rate(litres per day), and Me is COD of methane soluble in effluent at 35 °C (= 0.064 g/L) (van Haandel and Lettinga 1994).

[4] COD removed through sulfidogenesis = ∆SO42–

(COD) = 2Qe(Se – Si)/3

where Se is effluent SO42– concentration (grams per litre)

and Si is influent SO42– concentration (grams per litre).

Results and discussionsThe reactors were monitored continuously and the average

values based on three to four consecutive measurements at

pseudo steady state (PSS) have been reported. The temporalvariations in COD and sulfate removal performance for thereactors during study are given in Figs. 2a–2c. In general,the performance of reactors has been evaluated in terms ofprocess efficiency as characterized by COD removal, sulfateremoval, and biogas generation; and the reactor stability as

© 2004 NRC Canada

Patidar and Tare 423

evidenced by volatile fatty acid concentration, alkalinitylevel, and pH. Figures 3–5 depict a comprehensive pictureof the course of various performance parameters at PSS indifferent phases of the study. Micro-nutrients in the feed andreactors were monitored and their values were considered inassessing limiting concentrations (Table 2). Mass balance onCOD and sulfate is presented in Figs. 6–7. The followingsections describe general observations and comments thatare made on the basis of the results of various phases of thestudy.

Phase A: start up and operation without external sulfateand nutrients supplementation

This phase of the study involved start up and operationwithout external sulfate and nutrient supplementation. Thestart-up operation was completed on day 36, 76, and 76 forUASB, ABR, and HABR, respectively. All three systems

performed well with COD removal of more than 94%. Nonutrient limitation was observed, as available nutrients in thefeed contributed by ingredients might have been adequate tofulfill the requirements.

Phase B: sulfate addition without nutrientsupplementation

In this phase of the study, sulfate addition was started infeed to evaluate process performance for sulfate laden wasteand assess nutrient limitation, if any. Sulfate addition helpsin removal of COD by sulfate reducing bacteria, which uti-lize SO4

2– as an electron acceptor while metabolizing hydro-gen and acetate as substrate (eqs. [5] and [6]). Hydrogen andacetate are common intermediates of anaerobic degradationprocess.

[5] H2 + SO42– + H+ → HS– + 4H2O, ∆G0 = –38.1 kJ/mol H2

[6] CH3COO– + SO42– → HS– + 2HCO3

–, ∆G0 = –47.6 kJ/mol C2

During sulfate reduction, 1 mol of sulfate reduced acceptseight electrons, which is equivalent to electrons accepted by2 mol of O2. It means each mol of SO4

2– reduced removesCOD equivalent to 2 mol of O2 or 96 g of SO4

2– reduced re-moves 64 g of COD. Therefore, theoretically wastewaterwith COD:SO4

2– ratio of 64:96 (i.e., 0.67) contains enoughSO4

2– for complete COD removal through sulfate reduction.The COD:SO4

2– ratio lower than 0.67 indicates presence of

excess SO42– and extra substrate addition is necessary for

complete SO42– removal. On the other hand, wastes with

COD:SO42– ratio higher than 0.67 indicate limited SO4

2–

availability and methanogenesis is also required for com-plete COD removal. Sulfate reduction is associated withgeneration of sulfide, which causes direct toxicity tomethanogens as well as sulfate reducing bacteria and affectsnutrient availability due to precipitation of metals as metalsulfides. Therefore, sulfate reduction contributes positively

© 2004 NRC Canada

424 Can. J. Civ. Eng. Vol. 31, 2004

Fig. 2. Temporal variation in performance of reactors (a) UASB, (b) ABR, and (c) HABR.

for COD removal provided sulfide concentration remainsbelow toxicity level and nutrient availability is adequate. Inthis phase, soluble COD removal decreased to 65.82%,79.52%, and 77.04% in UASB, ABR, and HABR, respec-tively. Total COD removal has shown a similar decreasingtrend. Most of the COD removal was through methano-genesis as indicated by electron flow (≥88%) to MPB. Sul-fate removal of 88.5%–93% was comparable in the threesystems. Biogas methane content decreased to 45%–54% inthe three systems. Overall, sulfate addition and increase inOLR resulted in reduced COD removal in the three systems.Other possible reasons could be nutrient limitation and sul-fide toxicity. However, sulfide toxicity appears to be lesslikely reason because observed free H2S concentration(≤17 mg/L) is lower than reported inhibitory concentrationof 50 mg/L (Speece and Parkin 1983; Parkin et al. 1983;Kroiss and Wabnegg 1983). Rather low value of free H2Sconcentration is reported to be stimulatory, as it is one of thefour important nutrients for methanogens with optimum con-centration approximately 13 mg/L (Speece 1983). In UASB,significant biomass washed out in effluent during this phasethat increased sludge loading rate and is one of the reasonsfor poor COD removal compared with baffled systems. Onday 122, granulation was observed in UASB reactor andwash out might have been due to granulation process in

progress that involves selective removal of dispersed andlighter fractions of biomass and retention of heavier fractionfor growth of granules.

Phase C: performance evaluation at increased sulfateloading (COD:SO4

2– ratio of 6.9–7.0)In this phase, increase in sulfate loading caused perfor-

mance deterioration with COD removal efficiency as low as55.2%, 41.2%, and 52.4%, respectively for UASB, ABR,and HABR systems. A similar decreasing trend was ob-served for total COD removal. The concentration of VFA in-creased to 1.4, 1.7, and 1.5 g/L as acetic acid (HAc) inUASB, ABR, and HABR systems, respectively. Sulfate re-moval was 95.6%, 88.5%, and 92.4% in the three systems,respectively. Annachhatre and Suktrakoolvait (2001) ob-served 77% COD removal at COD:SO4

2– ratio of 6.66. Thisis relatively higher than that observed in the present phase ofthe study. This may be due to (i) nutrient limitation and(ii) sulfide toxicity. For the three systems, free H2S in efflu-ent (≤27.0 mg/L) was below toxicity level reported in theliterature to cause significant inhibition. Free H2S concentra-tion as low as 50 mg/L has been reported as inhibitory(Speece and Parkin 1983; Parkin et al. 1983; Kroiss andWabnegg 1983). Yamaguchi et al. (1999) suggested keepingfree H2S levels in reactor liquid below 100 mg/L for a satis-factory COD removal. Whereas, Visser et al. (1993) reported50% reduction in methanogenic activity while utilizing ace-

© 2004 NRC Canada

Patidar and Tare 425

Fig. 3. Soluble COD, total COD, and sulfate removal and efflu-ent dissolved sulfide at PSS during various phases. (SCOD =soluble COD; TCOD = total COD; DS = dissolved sulfide).

Fig. 4. Average gas production rate, effluent unionized H2S, CH4

content, and methane yield at PSS during various phases.

tate in thermophilic condition (55 °C) at pH levels of 6.3–8.0 and free H2S concentration of 18–24 mg/L.Maillacheruvu and Parkin (1996) have reported 110 mgsulfide/L and 625 mg sulfide/L as inhibition coefficients (Ki)for acetate-utilizing methane-producing bacteria (AMPB) andhydrogen-utilizing methane-producing bacteria (HMPB), re-spectively. It appears that the effect of sulfide toxicity mighthave been negligible keeping in view the low free H2S con-centration observed in the effluent and higher inhibition co-efficient of MPB. The effluent analysis for dissolved Fe, Ni,Zn, and Co at the end of the present phase indicated Ni andCo limitations, as their observed concentrations of 0.015–0.017 and 0.027–0.029 mg/L were lower than the nonlimitingconcentrations of 0.1–0.2 and 0.2–0.4 mg/L, respectively formethanogens (Speece et al. 1983; Parkin et al. 1990). Inview of this, it appeared that nutrient limitation was mainlyresponsible for poor COD removal in all three systems. Thelimitations of Ni and Co might have been due to precipita-tion by sulfide or higher requirement of Ni and Co toalleviate sulfide toxicity by excreting specific ligands by mi-croorganisms to bind sulfide. Barker and Stuckey (1999)pointed an increase in residual COD production as a typicaldefense mechanism by bacteria against toxic materials. Bar-ber and Stuckey (2000a) have shown that sulfide precipi-tated the micro-nutrients (especially Fe) present in ABR. Itwas calculated that approximately 35% of the sulfide gener-

ated was instantaneously precipitated as metal sulfide. Thelow concentration of micro-nutrients results in excessive re-sidual COD production that further lowers availability of Niand Co by complexion. Kuo (1993) speculated that solublemicrobial products (SMP) are produced in response to toxicsubstances and cited several other factors, that cause SMPproduction including (i) SMP production to scavenge whennutrients are available in low concentration and (ii) SMPproduction to relieve environmental stress such as tempera-ture changes, and osmotic shocks. Kuo and Parkin (1996)have shown that anaerobic microorganisms produce moder-ate concentration of nickel-chelating SMP of relatively lowstrength and moderate capacity when compared with simpleorganic compounds such as acetate, and citrate. They foundthat amount of SMP produced in a 40-d SRT chemostatcould chelate approximately 44 mg of Ni that is substantialconcentration. In the present study, it could not be con-cluded whether precipitation of nutrients by sulfide or higherrequirement of nutrients to avoid sulfide toxicity by specificligands was mainly responsible for nutrient limitation.

Phase D: performance evaluation at COD:SO42– ratio of

6.9–7.0 with nickel and cobalt supplementationThe previous phase of the study indicated the possibility

of nutrient deficiency as a cause for poor COD removal inall three systems. Therefore, in the present phase of thestudy performance was evaluated with supplementation oflimiting nutrients Ni and Co in the feed. Soluble and totalCOD removal in UASB increased from 55 to 94.5% and53.4% to 85.7%, respectively. It confirmed that mainly nutri-ent limitation was responsible for poor organic removal inthe UASB reactor. Increased gas production, methane con-tent, and electron flow to MPB showed improved methano-genesis after alleviating nutrient limitation. Whereas, solubleCOD removal in ABR and HABR improved slightly from41.2% to 45.9% and 52.4% to 56.1%, respectively. A similarmarginal increase was observed for total COD removal. De-crease in gas production and electron flow to MPB in ABRand HABR indicated inhibition of MPB. The yield of meth-ane decreased from 0.386 to 0.156 m3/kg COD removed at35 °C and 0.313 to 0.177 m3/kg COD removed at 35 °C, re-spectively, in ABR and HABR systems. It showed almost50% inhibition of methanogenesis. Sulfate reduction in allthree systems was ≥96.5% showing no inhibition of SRBdue to nutrient limitation. Dissolved sulfide concentrationwas comparable (132–137 mg/L) with low unionized H2S(≤25 mg/L) in effluent. Overall, on the basis of the results ofthe present phase of the study it can be stated that nutrientlimitation due to precipitation by sulfide significantly af-fected the process performance and supplementing limitingnutrient in the UASB system could restore the same. How-ever, it appears that partial phase separation in ABR andHABR aggravated the sulfide toxicity problem and contrib-uted to poor performance.

Phase E: performance evaluation at increased sulfateloading rate (COD:SO4

2– ratio of 5.76–5.84) with Znsupplementation

In the present phase of the study, performance was evalu-ated at increased sulfate loading with supplementation of Znin addition to Ni and Co in the feed because low dissolved

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426 Can. J. Civ. Eng. Vol. 31, 2004

Fig. 5. Effluent VSS, pH, electron flow to MPB, and electronflow to SRB at PSS during various phases.

Zn concentration was observed at the end of phase C. Noappreciable effect was observed on COD and sulfate re-moval in UASB system. However, improved COD removalwas observed in ABR and HABR. No significant effect onsulfate removal was observed as all three systems were oper-ating in sulfate limiting conditions with sulfate removal effi-ciency ≥95%. Further increase in removal was not possibleas effluent SO4

2– concentration was 19–34 mg/L ≤ KSO4(half-velocity coefficient for SO4

2–). Typical values of KSO4

range from 30–45 mg/L (Lens et al. 1998). Gas productionincreased because of improved COD removal. Overall,increase in SULR did not affect process performance ofUASB and ABR. However, it improved the COD removalrate in HABR. Also, no perceptible effect of Zn supple-

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Patidar and Tare 427

Nutrient concentration (mg/L)

Fe Ni Co Zn Mo

Phase UASB ABR HABR UASB ABR HABR UASB ABR HABR UASB ABR HABR —

C 0.500 0.550 0.42 0.015 0.016 0.017 0.028 0.03 0.028 — 0.061 — —E 0.430 0.744 0.252 0.288 0.302 0.303 0.021 0.05 0.058 0.190 0.118 0.572 —F 0.206 0.326 0.374 0.289 0.268 0.351 0.027 0.08 0.057 0.073 0.115 0.096 —G 1.574 0.284 0.371 0.332 0.390 0.308 — — — 0.290 0.297 0.229 —H 0.415 0.593 0.457 0.401 0.414 0.410 0.035 0.084 0.206 0.150 0.179 0.203 —I 0.418 0.427 0.521 0.376 0.376 0.449 0.293 0.365 0.367 0.170 0.136 0.196 —J 0.660 2.46 0.580 — — — 0.130 0.44 0.286 — — — —Feed* 0.48 0.075 0.11 0.179 0.073

*Without external nutrient supplementation.

Table 2. Nutrient concentration in feed and effluent during various phases of the study.

Fig. 6. Chemical oxygen demand mass balance in reactors(a) UASB, (b) ABR, and (c) HABR at PSS during variousphases. (Eff = effluent COD; CH4(g) = CH4(gas COD); CH4(aq) =CH4(aqueous COD); DSO4

2– = COD used in sulfate reduction; UA =unaccounted COD).

Fig. 7. Sulfate mass balance in reactors (a) UASB, (b) ABR, and(c) HABR at PSS during various phases. (Eff = effluent SO4

2–;Eff-DS = SO4

2– as effluent dissolved sulfide; H2S (gas) = SO42– as

H2S in gas; UA = unaccounted SO42–).

mentation was observed. Possibly the three systems were notZn limiting.

Phase F: performance evaluation at increased sulfateloading rate (COD:SO4

2– ratio of 4.43–4.67) with Mosupplementation

In phase F, performance was evaluated with increased sul-fate loading and supplementation of Mo in addition to Ni,Zn, and Co in the feed. The dose of Co was also increasedfrom 0.2 to 0.3 mg/L to ensure adequate availability. No ap-preciable effect on COD removal was found in UASB sys-tem. However, total COD removal decreased from 48% to24.5% in ABR system. The performance of HABR deterio-rated significantly as soluble and total COD removalreduced from 65.9% to 26.7% and 64% to 16.9%, respec-tively. The average gas production rate (AGPR) reducedfrom 1.53 to 0.22 m3/(m3·d) and CH4 content in biogas re-duced from 45.6% to 24.9%. However, sulfate removal wasstill very good with only slight decrease in the removal ratefrom 96.01% to 94.43%. It shows that most of the COD re-moval in HABR was through sulfate reduction. Possiblyopening of a HABR to remove excess sludge after cloggingmight have caused O2 shock and selective removal of at-tached biomass containing higher MPB population leadingto poor COD removal through methanogenesis. Other possi-ble reason could be increased sulfide toxicity due to increasein SULR. Dissolved sulfide (DS) concentration increasedmore than 200 mg/L and free H2S concentration increased to35–38 mg/L in the three systems. Severe inhibition tomethanogenesis observed in HABR, as shown by poor CODremoval, very low gas production with 25% CH4 content,and significant increase in electron flow (≈79%) to SRB.Overall, performance of UASB was excellent at increasedSULR. However, ABR showed slight decrease in COD re-moval with excellent sulfate removal. The hybrid anaerobicbaffled reactor exhibited severe inhibition of methano-genesis, possibly due to O2 shock, selective removal of MPBpopulation, and increased sulfide toxicity.

Phase G: performance evaluation at increased sulfateloading rate (COD:SO4

2– ratio of 3.06) with Fesupplementation

In the above-mentioned phase, the performance was eval-uated with increased sulfate loading and supplementation ofFe in addition to Ni, Zn, Co, and Mo in the feed. The possi-bility of controlling sludge loading rate (SLR) in UASB sys-tem by refilling washed out biomass at the beginning of thepresent phase of the study was also investigated. However,this attempt was unsuccessful as UASB system did not ac-cept refilled biomass and it was washed out again. The per-formance of UASB deteriorated considerably as soluble andtotal COD removal decreased from 91.6% to 48% and 89%to 46.1%, respectively. Sulfate reduction also decreased from97.1% to 59.6%. The process instability observed in UASBwas indicated by decreased pH from 7.5 to 7.1, increasedunionized H2S concentration from 37 to 51 mg/L, increasedVFA concentration to 1.41 g/L as HAc, bed lifting and heavybiomass washout. Possibly sulfide toxicity contributed toprocess instability and performance deterioration. SolubleCOD and sulfate removal were decreased from 46.3% to30.9% and 96.6% to 65.4%, respectively, in ABR system, in-

dicating SRB inhibition in the system. Gas production andCH4 yields were reduced, indicating increased inhibition ofMPB. Unionized H2S increased to 147 mg/L and DS to278 mg/L in ABR. In HABR system, soluble and total CODremoval improved slightly, however, sulfate removal de-creased from 94.4% to 65.7%. Gas production almostceased. The concentration of DS and unionized H2S in-creased to 238 and 90 mg/L, respectively, in HABR causinginhibition of both MPB and SRB. Electron flow to MPB de-creased from 81.8% to 60.3%, 30% to 16%, and 9% to 6%,respectively, in UASB, ABR, and HABR systems that indi-cates reduced COD removal through methanogenesis. TheABR and HABR have shown significant decrease in SO4

2–

removal. It was possibly due to increased free H2S concen-tration and toxicity to SRB. In this phase of the study, sig-nificant performance deterioration was observed in UASBsystem due to process instability causing sludge bed lifting,heavy biomass washout, and consequently increased SLR.The SEM examination of washed out biomass indicated hol-low granule formation in UASB (Fig. 8). Fe limitation andincrease in SULR and other factors might have promotedgrowth of bulky, fragile, and low-density hollow granules.Low Fe concentration (0.2–0.37 mg/L) was indicated byanalysis of effluent in the beginning of the phase. The roleof Fe is well documented in development of granular bio-mass. Sam-Soon et al. (1987) indicated importance of Fe inincreased extrapolymeric substances (EPS) formation by ty-ing up cysteine. Fe limitation may reduce EPS formation,and consequently weaker granule structure as EPS are re-ported to play an important role in granulation. In the pres-ent study, SULR has been increased through various phasesleading to change in substrate composition, cation concen-tration, and ionic strength; change in microbial consortia ingranules; and change in various properties of granules, suchas surface morphology, granular strength, density, and set-tling velocity. Sulfidogenic conditions favor growth ofweak, low density, and hollow granules due to increasedmass transfer limitation owing to prevailing conditions. Insulfidogenic system, low gas production results in low posi-tive effect of biogas release on decrease in diffusion resis-tance of surrounding liquid, consequently increasing masstransfer resistance (Huisman et al. 1990). Also, deposition ofsulfurous precipitates on a granule surface contributes to in-crease in substrate mass transfer resistance (Annachhatreand Suktrakoolvait 2001). Both these factors, in addition toincreasing the granule size, lead to the autolysis of cells atthe core of granules leading to formation of hollow granules.According to Beeftink and van den Heuval (1987) densitydiminishes with increasing diameters of granules. In theiropinion, this phenomenon is due to the substrate limitationat the center of large aggregates and subsequent autolysis oforganisms in the center to form hollow aggregate. Studieshave documented growth of granules with a somewhat lowergranular strength and density in sulfidogenic reactors (Lenset al. 1998). Change or shift in microbial consortia with in-creasing SULR may also be responsible for formation ofbulky, low density, and fragile granules. The MPB are re-ported to form dense granules by their specific morphology(e.g., Methanosaeta spp. (Wiegant 1988)) or by their hydro-phobic properties (Thaveesri et al. 1995). Soluble microbialproducts (SMP) or extracellular polymers (ECP) or extra-

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428 Can. J. Civ. Eng. Vol. 31, 2004

polymeric substances (EPS) are reported to play an impor-tant role in granulation. The ECP are important for structureand maintenance of granules. The composition of ECP af-fects the surface properties of the bacterial flocs and physi-cal properties of the granular sludge. The pore size andporosity of the ECP matrix affect cell activity through regu-lation of substrate and biomass transport. Low pore size andporosity may cause substrate transport limitation resulting inautolysis of the core of granules producing hollow granules.The production rate and type of ECP and (or) EPS and (or)SMP produced are reported to depend on many factors.These factors include substrate composition (Shen et al.1993a; Jia et al. 1996; Bal and Dhagat 2001; Schmidt andAhring 1994), composition of granules or microbial consor-tia (Jia et al. 1996), type of reactor (Barker et al. 1999), Feavailability (Sam-Soon et al. 1987), substrate strength, bio-mass concentration, organic loading, and solids retentiontime (Barker and Stuckey 1999). In view of above discus-sion, it appears that Fe limitation, increase in sulfate load-ing, and many other factors have played a key role in UASBprocess instability.

Phase H: performance evaluation at reduced organic andsulfate loading rate with nutrient supplementation

In this phase, the performance of UASB system improvedwith the increase in soluble COD, total COD, and sulfate re-moval from 48% to 63.9%, 46.2% to 57.4%, and 59.63% to96.6%, respectively. Biogas CH4 content and CH4 yield inUASB increased from 33.1% to 44.9% and 0.16 to0.21 m3/kg COD removal, respectively. However, unionizedH2S concentration of 83 mg/L observed in UASB was still atthe reported toxic level. Sulfate removal in ABR and HABRimproved from 65.4% to 97.5% and 65.7% to 80.5%, re-spectively. No appreciable change in COD removal was ob-served. Reduction in OLR had positive effect on CODremoval in the UASB reactor despite inhibitory free H2Sconcentration in the system. It appears that adequate avail-ability of nutrients coupled with reduced loading rateshelped in stabilizing the UASB process performance. How-ever, ABR and HABR did not show appreciable improve-ment as systems were stressed because of chronic toxicity

resulting from the pH gradient. The reduction in SULR alsohad positive effect as shown by significantly improved sul-fate removal in all three systems. In fact, improved sulfateremoval in the three systems was responsible for increasedCOD removal. Electron flow indicated 60% COD removalthrough methanogenesis and the remaining 40% through sul-fate reduction in UASB reactor. It shows that MPB and SRBwere competing for common substrates. Electron flow inABR and HABR indicated 70% COD removal throughsulfidogenesis.

Phase I: performance evaluation at increased sulfateloading rate (COD:SO4

2– ratio of 2.03) and Fe doseIn the above-mentioned phase, sulfate loading was further

increased with an increase in Fe dose to ensure its adequateavailability.The removal of soluble COD increased from63.9% to 71.6% but sulfate removal decreased from 90.6%to 78.3% in the UASB system. The increase in SULR andhigh unionized H2S (≈92 mg/L) appears to contribute to re-duced sulfate removal in UASB. Soluble COD and sulfateremoval reduced from 40.6% to 32.3% and 97.5% to 73.7%,respectively, in ABR because of increased sulfate loadingand consequently both MPB and SRB inhibited at high freeH2S concentration of 213 mg/L. Marginal improvement inCOD removal was observed in HABR. However, sulfate re-moval reduced from 80.5% to 72.4%. High DS and free H2Sconcentrations of 465 and 225 mg/L, respectively, were ob-served because of an increase in SULR. Electron flow toMPB reduced from 59.8% to 54.2% and 30.1% to 16%, re-spectively, in the UASB and ABR system that shows inhibi-tion of MPB. Electron flow to MPB was only 6% in HABR,indicating severe inhibition of MPB. Overall, sulfate re-moval performance of all three systems deteriorated becauseof increased SULR with marginal improvement in COD re-moval in UASB through sulfidogenesis.

Phase J: performance evaluation with supplementation ofnutrient combination Fe and Co

The investigations with batch assays by Patidar (2001) inparallel to reactor system study indicated maximum totalmethanogenic activity (TMA) and total sulfidogenic activity(TSA) stimulation in the assay supplemented with Fe andCo. Therefore, in this phase, the performance of reactorswas evaluated with supplementation of nutrient combinationFe and Co in feed. The removal of soluble COD, total COD,and SO4

2– improved from 71.6% to 78.5%, 57.8% to 72.8%,and 78.3% to 85.4%, respectively, in UASB system. BiogasCH4 content and CH4 yield also increased. The performanceof UASB improved despite DS and free H2S concentrationof 462 and 97 mg/L, respectively. A slight increase in CODremoval was observed in ABR system. However, sulfate re-moval decreased from 73.7% to 60.7%. Biogas CH4 contentand CH4 yield increased from 28.8% to 44.5% and 0.05 to0.094 m3/kg COD removal, respectively, with a marginal in-crease in AGPR. No appreciable change in soluble and totalCOD as well as sulfate removal was observed in HABR sys-tem. Increase in CH4 content, AGPR, and CH4 yield was ob-served but DS and free H2S concentration decreased from465 to 440 mg/L and 225 to 205 mg/L, respectively.Supplementation of Fe and Co combination resulted in im-proved methanogenesis as indicated by increased electron

© 2004 NRC Canada

Patidar and Tare 429

Fig. 8. Scanning electron micrographs of UASB washed outgranule (hollow granular structure with fragile irregular surface).

flow to MPB from 54.2% to 66.2%, 16% to 32.2%, and 6.2%to 16.2% in UASB, ABR, and HABR systems, respectively.COD removal through methanogenesis increased from 54%to 66% in UASB. Also in ABR and HABR, COD removalthrough methanogenesis increased to 32.2% and 16.2%, re-spectively. Overall, performance of systems did not deterio-rate in this phase; rather it showed stimulation particularly inUASB and ABR even after discontinuing supplementation ofNi, Zn, and Mo. It showed that systems were not Ni, Zn, andMo limiting but only Fe and (or) Co limiting. It supportedbatch assay study observations and indicated higher require-ments of Fe and Co.

Overview of results and discussions

Effect of sulfide on nutrient bioavailabilityThe present study clearly demonstrated that the addition

of sulfate and consequently generation of sulfide reducedmicro-nutrient availability because of precipitation as metalsulfide at a relatively low sulfide concentration, as observedin phase C. Literature stresses more on direct toxicity due tosulfide on MPB and SRB. However, nutrient limitation mayhave equally serious effect on activity of MPB and SRBeventually leading to process failure.

Effect of critical nutrients and their combinationsMany studies have indicated positive effects of nutrient

supplementation on process performance. However, most ofthem selected one or few of trace metals for the investiga-tion. The present study has shown that arbitrary selectionand supplementation of trace metals may not necessarily im-prove process performance. Rather, it will unnecessary in-crease the cost of treatment. Critical nutrient combinationsand their relative concentration requirement depend on bio-mass, operating conditions, and wastewater composition. Apilot plant or laboratory scale study for nutrient requirementwill provide adequate information for selecting and supple-menting critical nutrient(s) to achieve better process perfor-mance at low cost.

Effect of system configuration on nutrient requirementSystem configurations affect SMP production that affects

bioavailability of nutrients through complexation. However,in the present investigation SMP characterization could notbe done to conclude and comment about relative influenceof UASB, ABR, and HABR on nutrient availability.

Conclusions

The present investigations showed that process perfor-mance is greatly influenced by nutrient availability. Nutrientlimitation was observed at COD:SO4

2– ratio of 7.0 due toprecipitation by sulfide. The performance of UASB systemwas restored after supplementing limiting nutrients Ni andCo. However, baffled systems (ABR and HABR) did notshow appreciable improvement in process performancebecause of acute sulfide toxicity. Fe limitation and sulfido-genic conditions promoted growth of low density, fragile,hollow, and granular biomass leading to heavy washout andprocess instability. However, reduced loading rates andsupplementation of Fe with other nutrients helped in stabi-

lizing process performance. Supplementation of nutrientcombination Fe and Co improved COD and sulfate removalin UASB not withstanding withdrawal of Ni, Zn, and Mosupplementation during the last phase of study. It supportedbatch assay study observations and indicated higher require-ments of Fe and Co for maximum stimulating effect onTMA and TSA. The stimulating effect of nutrientsupplementation was most significant in UASB, as othersystems were stressed because of chronic and acute sulfidetoxicity.

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