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Renewable Energy 83 (2015) 455e462

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Renewable Energy

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Enzymatic hydrolysis and anaerobic biological treatment of fishindustry effluent: Evaluation of the mesophilic and thermophilicconditions

J.G. Duarte b, L.L.S. Silva a, D.M.G. Freire b, M.C. Cammarota a, *, M.L.E. Gutarra a, c

a Department of Biochemical Engineering, School of Chemistry, Federal University of Rio de Janeiro, Cidade Universit�aria, Centro de Tecnologia, Bl. E, Sl. 203,Ilha do Fund~ao, 21941-909 Rio de Janeiro, Brazilb Department of Biochemistry, Institute of Chemistry, Federal University of Rio de Janeiro, Cidade Universit�aria, Centro de Tecnologia, Bl. A, Sl. 549,Ilha do Fund~ao, 21949-900 Rio de Janeiro, Brazilc Federal University of Rio de Janeiro, Campus Xer�em, Estrada de Xer�em, 27, Xer�em, 25245-390 Duque de Caxias, Brazil

a r t i c l e i n f o

Article history:Received 23 July 2014Accepted 23 April 2015Available online 15 May 2015

Keywords:Anaerobic treatmentEffluentEnzymatic hydrolysisFish industryMesophilicThermophilic

* Corresponding author. Tel.: þ55 21 3938 7568; faE-mail address: christe@eq.ufrj.br (M.C. Cammaro

http://dx.doi.org/10.1016/j.renene.2015.04.0560960-1481/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

Enzymatic hydrolysis and anaerobic treatment of effluent similar to that generated in the fish processingindustry were evaluated at 30 �C and 50 �C. Hydrolysis used lipase produced by fungus Penicilliumsimplicissimum in solid state fermentation with babassu cake as substrate, which has optimal activity at50 �C. Hydrolysis kinetics was conducted with mixtures of effluent (containing 1500 mg oils and greases/L) and different lipase activities (0e0.67 U/ml of effluent), verifying that with 0.16 U/ml of effluent,9.69 mmol/ml of free acids were produced after 4 h at 50 �C. Anaerobic biodegradation assays wereconducted with effluent submitted to three different treatments: thermophilic (hydrolysis and anaerobictreatment at 50 �C), mesophilic (hydrolysis and anaerobic treatment at 30 �C) and hybrid (hydrolysis at50 �C and anaerobic treatment at 30 �C). The best results (97.5% of chemical oxygen demand [COD]removal and 105.4 ml CH4/g CODremoved) were obtained with the hybrid treatment in only 68 h. Thethermophilic hydrolysis not only reduced the amount of enzyme and the hydrolysis time but alsoreduced the time and the cost of mesophilic anaerobic treatment, favoring the application of thistreatment on an industrial scale.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Fish industry effluents have high levels of chemical oxygen de-mand (COD), temperature, oil and grease, and total suspendedsolids (TSS). They are generally discharged into water bodies afterreceiving treatment that is incompatible with their degree ofpollution, causing serious environmental damage [1].

Although aerobic processes are traditionally used in the treat-ment of industrial effluents, anaerobic processes are gainingprominence due to the advantages they bring, such as the removalof organic matter andmethane generation from high-load effluents(COD > 4000 mg/L), such as those produced by the fish industry[2e4].

x: þ55 21 3938 7567.ta).

However, anaerobic processes are adversely affected by the highoil and grease concentrations in these effluents, which can formagglomerates or pellets in the sludge flocs, hindering sludge sedi-mentation and reducing the efficiency of the treatment [1,5,6].

Alternatives to the pretreatment of effluents rich in fats toreduce their oil and grease content and aid the subsequent bio-logical treatment should therefore be investigated. One such po-tential alternative is enzymatic pre-hydrolysis with lipases(glycerol ester hydrolase, E.C. 3.1.1.3), enzymes that generally act inthe aqueouseorganic interface, catalyzing the hydrolysis of tri-acylglycerols [7e11].

Enzymatic pretreatment is away of improving the activity of themicrobial population in the subsequent biological treatment, sinceit prevents the accumulation of fats in the sludge. In addition, itpermits the conversion of complex organic compounds in the formof fats and proteins, which would be discarded as problematic solidwaste, intomethane, which can be used as a source of energy in thesame industry.

J.G. Duarte et al. / Renewable Energy 83 (2015) 455e462456

The application of these enzymes is growing steadily because oftheir ability to catalyze a wide range of reactions, including thehydrolysis of oils and greases in effluents from fish industry [7].However, for the enzymatic pre-hydrolysis step in effluent treat-ment to be economically feasible on an industrial scale, less costlyways of producing these enzymes, such as solid state fermentation(SSF), should be adopted.

SSF can use agroindustrial waste as a support and substrate forthe growth of microorganisms that not only reduce the total cost ofthe process, but are also biodegradable. Fermented solids con-taining enzymes called solid enzyme preparations (SEP), obtainedby SSF, can be used directly in the treatment of effluents, bypassingthe enzyme extraction and/or recovery steps. Thus, SSF assignsvalues to a residue that would be discarded into the environment,transforming it into a byproduct of great interest to various in-dustries [11,12].

This study evaluated the anaerobic biological treatment of a fishindustry effluent at 30 �C and 50 �C, with and without preliminaryhydrolysis at 30 �C and 50 �C, using a pool of enzymes rich inthermophilic hydrolase obtained by SSF.

2. Material and methods

2.1. Synthetic effluent

In view of the extreme variability of the composition of the in-dustrial effluent used in a prior study [27], we chose to use a syn-thetic effluent whose compositionwas similar to the industrial one.The synthetic effluent was prepared following a methodologyproposed by Roll�on [1] using 160 g heads, viscera, bones, and tails offresh sardines (Sardinella sp.) and 400 ml distilled water. Themixture was homogenized in a blender at the slowest speed for1 min, then sieved (1 mm average pore size) to obtain a concen-trate. An aliquot of this concentrate was removed to characterize itsCOD and oil and grease content, with the remainder being storedat �20 �C. At the time of use, the concentrate was diluted indistilled water to obtain the desired oil and grease concentration,and had its pH, COD, BOD5, total Kjeldahl nitrogen, total phos-phorus, oil and grease, and total solids determined.

2.2. Microorganism and propagation medium for the production ofenzymes

The enzyme preparation for use in the hydrolysis was producedusing a strain of Penicillium simplicissimum as an inoculum, selectedfor its capacity to produce lipases in different semi-synthetic me-diums [13] and in a medium composed of babassu cake [14]. Thestrain was propagated at 30 �C for 7 days in a medium with thefollowing composition (% w/v): soluble starch, 2.0; MgSO4.7H2O,0.025; KH2PO4, 0.05; (NH4)2SO4, 0.5; CaCO3, 0.5; yeast extract, 0.1;olive oil, 1.0; and agar, 2.5. After 7 days of growth, spores werescraped and suspended in a sterile phosphate buffer (50 mM, pH7.0), forming a spore suspension whose concentration was deter-mined by counting in a Neubauer chamber.

2.3. Solid state fermentation (SSF)

The raw material used as a fermentation medium was babassucake, a byproduct from the production of babassu oil (provided byTocantins Babaçu S.A.), which was supplemented with 6.25% (w/w)molasses (waste product from sugar production). The cake ob-tained was ground in a Wiley mill to yield particles with diametersof up to 3mm, although only particles of <1.18mmwere used in theproduction of the enzyme preparation. Fermentations were con-ducted in cylindrical tray-type reactors (15 cm height and 10 cm

diameter). Each tray contained 15 g of the babassu cake forming a1 cm-deep layer to achieve a good aeration and heat transfer be-tween the cake and the surrounding space. The reactors wereinoculated with 107 spores/g of dry babassu cake and incubated in achamber with controlled temperature (30 �C) and humidity (90%)for 72 h. Part of the fermented cake was sampled to quantify pro-tease and lipase activity, and the remainder e the solid enzymepreparation (SEP) e was stored under vacuum until use.

2.4. Submerged fermentation

Submerged fermentationwas performed in 1000ml Erlenmeyerflasks containing 240 ml of one of the two medium compositions.The synthetic medium (C:N ratio of 8.9:1) had the followingcomposition (w/v): 1% meat peptone, 0.5% yeast extract, 0.05%MgSO4, 0.05% KCl, 0.001% FeSO4$7H2O, 1% glucose, 0.3% olive oil,diluted in 0.1 M sodium phosphate buffer at pH 7.6 [13]. The sub-merged fermentation with babassu cake used 2.5% (w/v) babassucake with particle size <1.18 mm suspended in distilled water. Inboth fermentations, the medium was inoculated with2 � 104 spores/ml, and the Erlenmeyer flasks were incubated in ashaker at 170 rpm and 30 �C [15]. Fermentation was monitored bymeasuring lipase activity, and was discontinued after 96 h. Thefermented broth was filtered throughWhatman filter paper (11 mmpore size) for biomass separation. An aliquot was used to determinelipase and protease activity, and the remainder (liquid enzymepreparation) was stored at 4 �C until use.

2.5. Enzymatic hydrolysis

Enzymatic hydrolysis was carried out in 500 ml Erlenmeyerflasks containing 250 ml synthetic effluent with an initial concen-tration of around 1500 mg oil and grease/L, agitated in a shaker at150 rpm andmaintained at 50 �C (thermophilic hydrolysis) or 30 �C(mesophilic hydrolysis). Initial tests were conducted with lipaseactivity of 0.67 U/ml of effluent at 50 �C with and without theaddition of 1% (w/v) sodium azide, an anti-microbial substance thatacts as a respiratory chain uncoupler. The azide was added to pre-vent the growth of microorganisms and the consumption of thefree acids produced so as to distinguish between the action ofenzymatic catalysis and that of microbial biodegradation. The ki-netics of the thermophilic hydrolysis was evaluated by determiningthe production of free acids over time by withdrawing 10 ml ali-quots every 4 h up to 24 h. Having selected the best hydrolysiscondition (at 50 �C), this was repeated without the addition ofsodium azide for subsequent use in anaerobic biodegradabilitytests. Mesophilic hydrolysis was conducted for 8 h, as described byAlexandre et al. [7], for subsequent use in anaerobic biodegrad-ability tests.

2.6. Anaerobic biodegradability tests

The enzymatically pretreated effluent and the crude effluent(without enzymes) were used in the anaerobic biodegradabilitytests. The tests were conducted in 100 ml penicillin flasks with90 ml working volume of a mixture composed of anaerobic sludgeand raw or pretreated effluent. The amount of sludge used in eachbiodegradability test was calculated to maintain an initial effluentCOD-to-sludge volatile suspended solids (VSS) ratio of 1:1. Theflasks were sealed with rubber stoppers and aluminum seals andincubated at 30 �C or 50 �C up to biogas volume stabilization.

The sludge was collected from a mesophilic upflow anaerobicsludge blanket reactor in operation at a poultry processing plant,and was adapted to the synthetic effluent simulating the effluentgenerated in fish industry. Sludge adaptation was conducted at

Table 1Characterization of synthetic effluent and industrial effluent.

Parameter Synthetic effluent Industrialeffluent [27]

pH 6.5e6.9 5.5e7.2COD (mg/L) 6000e15,767 1313e12,333BOD (mg/L) 2122 967e2900Total Kjeldahl nitrogen (mg/L) 123.3 5.3e499Total phosphorus (mg/L) 56.8 51e1400Oil and grease (mg/L) 2111e6317 78e3656Total solids (mg/L) 2271 848e1485Total fixed solids (mg/L) 208 140e230Total volatile solids (mg/L) 2063 618e1345

Table 2Production of free acids in the enzymatic hydrolysis of the synthetic effluent, con-taining 1500 mg oil and grease/L and different solid enzymatic preparation (SEP)concentrations with lipase activity of 62 U/g after 4 h (DT4) and 24 h (DT24) of hy-drolysis at 50 �C. The control experiments were conducted with effluent without theaddition of SEP.

Condition Free acid production(mmol/mL)a

DT4 DT24

Test 1 e Hydrolysis with and withoutSEP and sodium azide

Effluent without SEP (control) 2.57 8.34Effluent þ 0.67 U/mlb 17.81 26.86Effluent þ 0.67 U/mlb þ 1% (w/v) azide 16.02 25.93Test 2 e Hydrolysis with different levels

of lipase activityEffluent without SEP (control) 3.50 7.30Effluent þ 0.16 U/mlb 9.69 12.02Effluent þ 0.32 U/mlb 16.43 19.59Effluent þ 0.67 U/mlb 17.81 26.86

a DTt ¼ Ct � C0, where Ct ¼ concentration of free acids at time t;C0 ¼ concentration of free acids in the initial time (data not shown).

b U/ml of effluent. Lipase activity obtained by addition of different quantities ofSEP containing average lipase activity of 62 U/g.

J.G. Duarte et al. / Renewable Energy 83 (2015) 455e462 457

30 �C for mesophilic tests and 50 �C for thermophilic tests. For this,approximately 1/3 of the working volume of 1 L bottles was filledwith sludge, and were then topped up with a low-fat-contentsynthetic effluent (COD 300 mg/L). The bottles were sealed withrubber stoppers with a single outlet for the collection and mea-surement of the biogas produced by liquid displacement, andincubated at 30 �C or 50 �C. When biogas production stabilized(which took anything from 3 to 7 days), the supernatant wasreplaced by fresh diluted effluent, whose COD was graduallyincreased up to a maximum of 2000 mg/L.

The sludge was characterized for its total volatile solids content(TVS e 12,000 mg/L) and specific methanogenic activityðSMA e 0:173 g CODCH4

=g VSS:dÞ. SMA can be defined as themaximum methane production capacity of a consortium of anaer-obic microorganisms held under controlled conditions. It wasdetermined in 100ml penicillin flasks, with 10% headspace, withoutstirring, at 30 �C for 14 days, with an initial anaerobic sludge con-centration of 2 g TVS/L and a substrate (mixture of volatile organicacids e acetic, propionic and butyric) with 2.0 g COD/L, accordingChernicharo [3].

Biodegradability was measured by COD removal efficiency andbiogas production, as assessed by the displacement of the plungerof 20 ml graduated plastic syringes connected to the flasks until thecomplete stabilization of biogas production. After stabilization, thevolume of biogas in all the syringes was measured and the totalvolume of each flask was used to calculate the biogas. The biogasproduced was collected and injected into a Varian Micro GC 4900chromatograph to determine the percentage of methane. The flaskswere then opened to remove samples for quantification of pH andfinal COD. Each condition was evaluated in five replicates and theresults are presented as means and standard deviations.

2.7. Analytical methods

All the analyses required to characterize the effluent and sludgeand monitor the biodegradability tests were performed asdescribed in the Standard Methods [16]. Lipase activity wasmeasured by a spectrophotometric method based on the formationof a chromophore product obtained from the lipase-catalyzed hy-drolysis of p-nitrophenyllaurate [15]. One lipase unit was defined asthe amount of enzyme that released 1 mmol of p-nitrophenol underassay conditions. Protease activity was measured by spectropho-tometry as described by Bendicho et al. [17]. One protease activityunit was defined as the unit difference in absorbance between thereaction blank and the sample per minute under assay conditions.The production of free fatty acids during hydrolysis was monitoredin an automatic titrator (end point pH 11) using 0.04 MNaOH as thetitrant.

3. Results and discussion

3.1. Effluent characterization

The synthetic effluent was prepared with viscera and parts ofheads, tails and bones of Sardinella sp., as fishes' heads and visceracontain the highest oil and fat content [18]. Due to the presence ofblood, mainly from the heads, the synthetic effluent had effluenthad a reddish color and high COD and oil and grease values, as canbe seen in Table 1. Nevertheless, except for total volatile solids(TVS), these effluent's values fell within the ranges obtained for thefish industry effluents. The relatively high TVS concentration of thesynthetic effluent was due to the inclusion of virtually all the sus-pended material produced when it was made, since sievingremoves only very coarse solids, while in industrial effluents, suchmaterial is separated during fish processing.

3.2. Enzymatic hydrolysis

SSF with babassu cake results in enzymatic preparations withvarying lipase activities that yield different free fatty acid levels inthe hydrolysis step [19]. Thus, in order to compare the results ob-tained for the synthetic and industrial effluent, hydrolysis assayswere conducted with different amounts of enzyme preparation,measured by lipase activity per volume of effluent. Pre-hydrolysisof a fish industry effluent containing 1500 mg oil and grease/Lwith 0.2%, 0.5%, and 1.0% (w/v) P. simplicissimum solid enzymepreparation (SEP) with lipase activity of 134 ± 7 U/g at 30 �C wasmonitored for up to 18 h. The hydrolysis condition that resulted inthe highest methane volumes in the subsequent anaerobic treat-ment step was the one that used 0.5% SEP (or 0.67 U/ml of effluent)for 8 h of hydrolysis with an output of 4.7 mmol/ml of free acids [20].Thus, initial enzymatic hydrolysis tests (Test 1) were run at 50 �C,with and without the use of sodium azide, maintaining lipase ac-tivity of 0.67 U/ml of effluent. The results are shown in Table 2.

There was an increase in the concentration of free fatty acidsproduced by the action of the SEP in both conditions (with andwithout azide) and for both reaction times. Furthermore, it wasobserved that similar concentrations of free acids were produced in24 h by 0.67 U/ml of effluent with or without azide, indicating thatits use was not necessary for determining the optimal time andenzyme activity for anaerobic biodegradability tests. Differing fromthe findings of Alexandre et al. [7], in the hydrolysis conducted at30 �C, a decline in the free acid content was observed due to the

Fig. 1. Biogas production (50 �C, 1 atm) after different contact times between effluentand anaerobic sludge in the control experiments (non-hydrolyzed effluent) and witheffluent previously hydrolyzed with 0.16 or 0.32 U/ml of effluent, with both hydrolysisand anaerobic treatment conducted at 50 �C.

J.G. Duarte et al. / Renewable Energy 83 (2015) 455e462458

aerobic microorganisms'use of the acids produced as a substrate.This behavior was not observed in experiments carried out in thepresence of sodium azide, where the acid content continued toincrease throughout the assay [7].

As the synthetic effluent consisted only of water and sardines,most microorganisms were part of the fish biota. The parts used toproduce the effluent were responsible for most of the bacterialflora, which is usually concentrated in the intestines and gills of fish[21]. The sardines used in the synthetic effluent were from theBrazilian coast, i.e., fish from tropical waters with moderate tem-peratures. Most of the bacteria present in these organisms weretherefore mesophilic, which explains the low microbial activityobserved during hydrolysis at 50 �C.

The concentrations of free acids obtained after 4 h hydrolysis at50 �C (17.81 and 16.02 mmol/ml for the SEP with and without azide)were higher than those obtained by several authors under theirbest treatment conditions and highest subsequent methane pro-duction, which indicates that lipase activity in hydrolysis at 50 �Ccould be reduced to achieve lower concentrations of free fatty acidsand thus better results.

Therefore, further hydrolysis tests were conducted with 0.32and 0.16 U/ml of effluent by measuring the concentration of freeacids up to 24 h (Test 2). The results obtained at 4 and 24 h of hy-drolysis can be seen in Table 2.

Using 0.16 U/ml of effluent, the production of free acids was9.69 mmol/ml after 4 h of hydrolysis at 50 �C. In a previous study, amaximum variation of 7.3 mmol/ml of free acids was obtained after22 h hydrolysis at 35 �C with 1% w/v (0.21 U/ml) of the enzymepreparation from P. restrictum in the treatment of poultry slaugh-terhouse effluents with 1200mg oil and grease/L [11]. Hydrolysis ofeffluents from the dairy industry with 1000mg oil and grease/L and0.1% w/v (0.02 U/ml of effluent) of the same enzyme preparationresulted in an 8.0 mmol/ml increase in the production of free acidsafter 24 h of hydrolysis at 35 �C [8]. In other study, 0.5% w/v of theenzyme preparation from P. simplicissimum (0.67 U/ml of effluent)resulted in 4.7 mmol/ml after 8 h hydrolysis at 30 �C [20]. All theseresults were reported as the best conditions for the production ofmethane in anaerobic treatment subsequent to hydrolysis. Basedon the free acid concentrations, which ranged from 4.7 to 8 mmol/ml for various times and types of effluents, it was found that thebest activity to be used in hydrolysis prior to anaerobic biode-gradability tests would be 0.16 and 0.32 U/ml of effluent and 4 hhydrolysis, since this time showed the highest yield.

3.3. Anaerobic biodegradability tests in sequencing batches afterhydrolysis

Fig. 1 and Table 3 show biogas production over time and theresults obtained in anaerobic biodegradability tests, respectively,using three sequential batches with non-hydrolyzed effluent(control) and effluent previously hydrolyzed with 0.16 or 0.32 U/mlof effluent, with both hydrolysis and anaerobic treatment con-ducted at 50 �C. In the first and third batches, biogas productionstabilized after 72 h, while in the second batch it took 96 h tostabilize. This time is much shorter than the 12 days previouslyreported for complete biogas production stabilization at 30 �C [20].There was no lag phase for any of the three batches, indicating thatthe sludge was well adapted to the synthetic effluent's constituentsand to a temperature of 50 �C (data not shown).

Data presented in Table 3 show that in the first contact of thesludge with effluent containing 1500 mg oil and grease/L, similarCOD removal rates and methane percentages were obtained underall the conditions. The second contact of the sludge with theeffluent yielded a sharp drop in the biogas volume in the control(reduction of 71%), contrasting with what occurred when the

effluent was pretreated with 0.16 and 0.32 U/ml of effluent, wherethe reductions in the biogas volume were smaller (20% and 3%).This result was expected, since in the control condition there was amuch higher adsorption of non-hydrolyzed fats in the microbialbiomass, which impaired the metabolism of the organic matter inthe effluent [1], resulting in a 14% reduction in COD removal. Whenthe effluent was hydrolyzed, although there was an accumulationof non-hydrolyzed fats and a decrease in COD removal, it wasconsiderably lower (9% and 8%).

In the third batch, although biogas production was lower underthe control conditions, it actually decreased under all three con-ditions evaluated. One explanation for this sharp drop in biogasproduction is that the accumulation of fat exceeded the adaptationcapacity of the sludge, hampering the activity of microorganisms

Table 3Results of anaerobic biodegradability tests with raw effluent (control conditions) and effluent after hydrolysis with 0.16 and 0.32 U/ml of effluent, both at 50 �C.

Condition CODi (mg/L) CODf (mg/L) CODremoval (%)

Biogasvolume (ml)a

% CH4 SMP(ml CH4/g CODremoved)a,b

1st batchControl 6580 ± 10 284 ± 8 95.3 67.3 ± 5.8 74.0 ± 0.3 880.16c 7220 ± 200 318 ± 10 95.0 92.5 ± 10.0 68.0 ± 0.2 1010.32c 8065 ± 250 341 ± 10 95.2 86.0 ± 9.0 72.0 ± 0.2 892nd batchControl 5865 ± 10 1040 ± 5 82.3 19.8 ± 1.0 43.5 ± 0.1 200.16c 7780 ± 110 1045 ± 10 86.6 74.3 ± 4.5 93.0 ± 0.1 1140.32c 8600 ± 100 1060 ± 10 87.7 83.5 ± 4.7 82.0 ± 0.2 1013rd batchControl 7845 ± 15 2454 ± 20 68.72 4.3 ± 1.5 33.3 ± 0.5 30.16c 7590 ± 210 1972 ± 35 74.02 9.3 ± 3.5 53.7 ± 0.7 100.32c 7770 ± 160 2440 ± 25 68.60 9.5 ± 0.5 40.4 ± 0.4 8

a At 50 �C/1 atm.b Specific methane production.c U/ml of effluent, with SEP containing average lipase and protease activity of 75 U/g and 70 U/g at 50 �C.

J.G. Duarte et al. / Renewable Energy 83 (2015) 455e462 459

in COD removal and methane production. Contrasting behaviorwas observed at 30 �C, as the batches were conducted with themicrobial biomass in adaptation and increasing methane produc-tion [11].

Another explanation is that the proteases present in the SEPwere acting to reduce the microbial biomass. The reduction in thesludge concentration was greater in the presence of proteasessecreted by Brevibacillus sp. and minimized by the use of proteaseactivity inhibitors [22].

The high temperature is another factor that may have affectedmethane production, although the sludgewas previously adapted to50 �C. Hydrolysis at 50 �C may have released long-chain fatty acidsinto the medium, known inhibitors of methanogenic archaea [23].

3.4. Effect of proteases on anaerobic biodegradability

In order to assess whether the presence of proteases in the SEPwas harmful in the subsequent stage of methane production,enzyme preparations derived from three different fermentationsystems were used: submerged fermentation with synthetic me-dium, submerged fermentation with babassu cake, and solid statefermentation (SSF). Each of these enzyme preparations haddifferent levels of protease activity with respect to lipase activity.The ratio of enzyme activity (protease: lipase) was higher in the SEPobtained from SSF (1.16), intermediate in the liquid enzyme prep-aration obtained from submerged fermentation with babassu cake(0.4), and null in the liquid enzyme preparation from SSF. Thus,maintaining lipase activity at 0.16 U/ml of effluent for all theenzyme preparations evaluated, hydrolysis was carried out at 50 �Cfor 4 h. The hydrolyzed effluents were then submitted to anaerobicbiodegradability tests at 50 �C. The results are shown in Table 4.

In a previous biodegradability test (Table 3), three sequentialbatches were performed and a significant decrease in the param-eters evaluatedwas observed for the third batch. In this experiment(Table 4), two sequential batches were performed, followed by oneweek of sludge incubation at 50 �C, and two more sequentialbatches. The sludge incubation period was designed to assist in thesolubilization/hydrolysis of fats adhered to the biomass. In general,the COD removal was similar for all the conditions, dropping alongthe first three batches (90.5 ± 2.4, 86.6 ± 4.3, 74.2 ± 4.9%) andstabilizing in the fourth batch (79.2 ± 4.9%). Thus, differencesamong the conditions can best be evaluated by specific methaneproduction, which, under the control condition, yielded muchlower values in the first three batches and recovered in the fourthbatch, probably due to the adaptation of the microorganisms,which started producing and excreting hydrolytic enzymes in the

reactionmedium, and also due to the sludge recovery period, whichenabled the assimilation of fat accumulated before the thirdeffluent exchange.

Overall, the differences between one hydrolysis condition andanother were not great enough to justify the use of the enzymepreparation produced by SSF without proteases. Even so, the hy-drolysis condition with the highest protease activity (SSF) did nothamper the anaerobic treatment. Interestingly, fish industry efflu-ents contain a significant portion of biodegradable organic matterprimarily composed of proteins and lipids [6,24]. Thus, it isimportant to have proteases present in the hydrolysis of proteins inthe effluent; the proteases present in the enzyme prepared frompig pancreas help the treatment of effluents rich in fats andproteins [25].

The hydrolysis condition with 0.16 U/ml of effluent using theSEP from SSF for 4 h at 50 �C was selected to continue the work.Although the specific methane productionwas very low (comparedwith the theoretical value of 414 ml CH4/g CODremoved at50 �C/1 atm), this condition reduced treatment costs. In a previousstudy, the best condition obtained in the tests e 8 h hydrolysis at30 �C with 0.5% (w/v) SEPe yielded 216ml CH4/g at 30 �C [20]. Thisvalue corresponds to 230 ml CH4/g CODremoved at 50 �C, orapproximately 56% of the maximum expected theoretical value.This difference may be due to the higher temperature used in thehydrolysis and biodegradability tests, or because the effluent usedin this workwas synthetic, while in the previous study an industrialeffluent was used.

3.5. Anaerobic biodegradability test under mesophilic andthermophilic conditions after hydrolysis at 50 �C

The high energy consumption involved in maintaining thetemperature at 50 �C for anaerobic treatment may limit the feasi-bility of the process on an industrial scale. However, using thistemperature for only 4 h of hydrolysis may be feasible, sinceindustrial effluents are generated at high temperatures. Thus, hy-drolysis at 50 �C followed by anaerobic treatment at 30 �C and 50 �Cwas evaluated.

To carry out the anaerobic biodegradability tests, sludge previ-ously adapted to thermophilic and mesophilic conditions was sub-mitted to three consecutive batches with effluent hydrolyzed at50 �C for 4 h or 30 �C for 8 h (condition used in Ref. [20]) to obtainCODremoval over 80%. Thus, thebiogas production results under thethree conditions (thermophilic, mesophilic and hybrid) shown inFig. 2 correspond to the fourth contact of the sludgewith the effluentcontaining 1500 mg oil and grease/L hydrolyzed in each condition.

Table 4Results of anaerobic biodegradability tests with raw effluent (without addition of enzyme e control) and with effluent after hydrolysis with 0.16 U/ml of different fermen-tations, both at 50 �C.

Enzimaticpool (P:L)a

CODi (mg/L) CODf (mg/L) CODremoval (%)

Biogasvolume (mL)b

% CH4 SMP(ml CH4/g CODremoved)b,c

1st batchNo addition 3723 ± 40 307 ± 5 91.8 14.3 ± 3.5 85.1 ± 0.1 40SFd(0) 4267 ± 64 440 ± 12 89.7 87.0 ± 8.4 88.9 ± 0.2 225SFBe(0.4) 3477 ± 207 433 ± 48 87.5 43.3 ± 12.2 89.3 ± 0.1 141SSFf(1.16) 5107 ± 57 359 ± 14 93.0 97.5 ± 6.4 86.6 ± 0.1 1982nd batchNo addition 5463 ± 200 615 ± 30 88.7 46.0 ± 7.1 89.5 ± 0.05 94SF (0) 4673 ± 74 606 ± 31 87.0 62.8 ± 8.1 89.3 ± 0.06 153SFB (0.4) 3917 ± 112 767 ± 19 80.4 41.9 ± 13.7 89.5 ± 0.03 132SSF (1.16) 5907 ± 337 582 ± 3 90.1 60.7 ± 5.5 85.9 ± 0.04 1093rd batchNo addition 4552 ± 69 1219 ± 81 73.2 8.0 ± 0 82.9 ± 0.07 22SF (0) 6433 ± 115 1998 ± 30 68.9 50.5 ± 6.8 80.1 ± 0.09 101SFB (0.4) 4373 ± 326 1147 ± 32 73.8 56.5 ± 1.9 84.4 ± 0.05 164SSF (1.16) 6075 ± 171 1174 ± 60 80.7 80.5 ± 1.0 81.3 ± 0.01 1484th batchNo addition 5822 ± 42 1199 ± 14 79.4 66.5 ± 4.8 84.8 ± 0.07 136SF (0) 6372 ± 25 1656 ± 18 74.0 61.3 ± 3.1 85.2 ± 0.56 123SFB (0.4) 5128 ± 67 1151 ± 5 77.6 52.0 ± 2.9 85.3 ± 0.09 124SSF (1.16) 6517 ± 85 931 ± 7 85.7 68.5 ± 6.4 84.4 ± 0.02 115

a P ¼ protease; L ¼ lipase.b At 50 �C/1 atm.c Specific methane production.d SF ¼ submerged fermentation.e SFB ¼ submerged fermentation with babassu cake.f SSF ¼ solid state fermentation.

J.G. Duarte et al. / Renewable Energy 83 (2015) 455e462460

It was found that in the hybrid and thermophilic treatments,biogas production stabilized after around 68 h (2.8 days), whileunder mesophilic conditions it took about 180 h (7.5 days) tostabilize. In another study, biogas production took 12 days to sta-bilize under the same conditions [20], which is probably related tothe use of a more complex industrial effluent. The higher biogasyield under the hybrid conditions (376.1 ml biogas/L.d) was owingto the maintenance of a high initial production rate (51.1 ml/d) forup to 18 h, while under mesophilic conditions the yield was muchlower (139.4 ml biogas/L.d) because the initial production rate

Fig. 2. Biogas production (STP) over time for effluent with 1500 mg oil and grease/L submtreatment at 30 �C), thermophilic (hydrolysis and anaerobic treatment at 50 �C), and hybri

(49.0 ml/d) dropped as of 22 h and remained at an average of5.3 ml/d until it stabilized after 180 h. The thermophilic conditionsdid not produce good results, yielding only 20.9 ± 1.2 ml biogas, or18.8 ml methane.

Under the hybrid conditions, despite the considerably shorterhydrolysis time (only 4 h), the temperature used increased thehydrolysis reaction speed, allowing the fats present in the effluentto break down into free fatty acids and glycerol (which are moreeasily degraded by the microbial consortium) in a shorter space oftime, accelerating the subsequent anaerobic biological treatment

itted to hydrolysis/anaerobic treatment under mesophilic (hydrolysis and anaerobicd (hydrolysis at 50 �C and anaerobic treatment at 30 �C) conditions.

Fig. 3. Anaerobic biodegradability tests with effluent containing 1500 mg oil and grease/L submitted to hydrolysis/anaerobic treatment under hybrid conditions: specific productionof methane (STP) and COD over time.

J.G. Duarte et al. / Renewable Energy 83 (2015) 455e462 461

process. Moreover, the anaerobic digestion process is facilitatedwhen treatment occurs after thermophilic hydrolysis [26].

Fig. 2 shows that in the first 24 h of degradation, the effluentshydrolyzed at 30 �C and 50 �C maintained the same biogas pro-duction rate (about 2.3 ml/h). However, the effluents hydrolyzed at30 �C yielded a gradually declining biogas production rate untilstabilization at 180 h, while the effluents hydrolyzed at 50 �Cmaintained the same biogas production rate for 20 h and then sta-bilized. This behavior indicates thathydrolysis at 50 �C releasesmoreproducts capable of being assimilatedmore quickly by themicrobialpopulation, which is of the utmost importance for the anaerobictreatment process. The hybrid condition yielded the best biogasproduction results: 106.0 ± 1.7 ml, and 95.5 ml methane. Thus, thehigher hydrolysis temperature yielded biogas production rates ashigh as those obtained undermesophilic conditionse 104.1± 6.2mlof biogas or 93.8 ml methane e but in a much shorter time.

A comparison of the specific methane production (SMP) atstandard temperature and pressure (STP) under the three condi-tions revealed that the thermophilic conditions yielded SMP of19.5 ml CH4/g CODremoved, far below the theoretical value of350 ml CH4/g CODremoved at STP. This could be because there was aconcentration of methanogenic archaea adapted to 50 �C in thesludge collected from the reactor operating under mesophilicconditions. Under mesophilic conditions, the highest SMP of125 ml CH4/g CODremoved was obtained after 180 h. A fish industryeffluent yielded 194.6 ml CH4/g CODremoved under mesophilicconditions [20]. This indicates that the industrial effluent maycontain components that aid the conversion of organic materialinto methane.

The maximum SMP under hybrid conditions (Fig. 3) was110.6 ml CH4/g CODremoved after 228 h. However, in only 68 h it hadreached 105.4 ml CH4/g CODremoved, differing from the mesophilicconditions, under which it took 96 h to attain values close to themaximum (98.6 ml CH4/g CODremoved). As to COD removal, in only4 h of test, residual COD reached 25% of the original, which meansCODwas reduced by 75%. By 28 h, COD removal had reached 93.3%;in 116 h, it reached 97.9%, and by 228 h it had reached 98.2%. After68 h, COD removal reached 97.5% and SMP reached levels close tothe maximum obtained at the time of the test. Thus, 68 h is longenough for the effluent to reach the desired levels.

These results suggest that the factor limiting the reduction ofthe treatment time is not thermophilic anaerobic biodegradability,but thermophilic hydrolysis. The latter probably promotes therelease of components that aid the biodegradation of the effluentand methane production, while reducing the quantity of enzyme

required [26]. Hydrolysis at 50 �C reduces enzyme activity and re-action time and may be the key to accelerating the subsequentmesophilic anaerobic biological treatment, which, conducted atintermediate temperatures, would not increase process costs.

Anaerobic treatment can be conducted at room temperature inmost parts of Brazil because high temperatures can be guaranteedmost of the year. Even if the hydrolysis temperature has to bemaintained, the higher energy consumption could possibly beoffset by the shorter treatment time, which makes it an importanttarget for the scaling up of the enzymatic/biological process.

4. Conclusions

Theenzymatichydrolysis of fatspresent in the effluent at 50 �C for4 h led to concentrations of free acids that were compatible with thesubsequent anaerobic biological treatment and with smaller quan-tities of enzyme (0.32 and 0.16 U/mL of effluent) than the hydrolysisconducted at 30 �C. When hydrolysis and anaerobic biologicaltreatment were both conducted at 50 �C, it was found that thesmaller quantity of enzyme evaluated (0.16 U/mL of effluent) yieldedhigher specific methane production than in the control assays(without hydrolysis). Even so, the results obtained under thermo-philic conditions were lower than those obtained at 30 �C.Whenweevaluated the hybrid conditions, with hydrolysis at 50 �C andanaerobic biological treatment at 30 �C, the results obtainedwere thesame as those obtained when both stages were run at 30 �C, but in amuch shorter time (68 h). This indicates that these conditions (hy-drolysis at 50 �C and biological treatment at 30 �C) could be adoptedfor industrial scale combined (enzymatic/biological) treatments.

Acknowledgments

This work was supported by project funds from the BrazilianNational Council for Research and Development (CNPq) Process no557194/2010-5, Carlos Chagas Filho Foundation for Research Sup-port in the State of Rio de Janeiro (FAPERJ) Process no E-26/100.647/2007, and Petrobras Contract no 0050.0045686.08.2.

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