Ecological clarification of cheese whey prior to anaerobic digestion in upflow anaerobic filter
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plements can be produced from whey (Cristiani-Urbinaet al., 2000; Perle et al., 1995). Nevertheless, cheese produc-ing units usually do not proceed with investments for
and so a large number of dairies in dispose of their waste,
developing countries, which are confronting rapidlyincreasing water pollutions problems, anaerobic digestionbecomes even more favourable and promising (Agrawalet al., 1997; Shigeki and Hideki, 1999).
In Tunisia, anaerobic digestion of several industrialwastes has been carried out including tuna processing
* Corresponding author. Tel.: +216 71703627; fax: +216 71704329.E-mail address: firstname.lastname@example.org (M. Hamdi).
Available online at www.sciencedirect.com
Bioresource Technology 99 (2The dairy industry, like most other agro-industries, gen-erates residues of which whey is the most important waste-waters produced with extremely high organic content.Cheese whey (CW), a by-product of the dairy industry, itcontains a signicant amount of carbohydrates (45%),mainly lactose, proteins not exceeding 1%, fats at about0.40.5%, lactic acid less than 1%, salts that may rangefrom 1% up to 3% (Gelegenis et al., 2007). Several possibil-ities have been assayed for whey exploitation over the last50 years. Many useful food products such as protein con-centrates, yeast, lactose, lactic acid, and various feed sup-
especially cheese whey, into the environment in enormousquantities. This causes serious pollution problems sincewhey has a high heavy organic pollutant with high chemi-cal oxygen demands (COD) (6080 g/l) (Mockaitis et al.,2006). 97.7% of total COD of the wastewater wasaccounted for by lactose, lactate, protein and fat (Hwangand Hansen, 1998). Extremely high organic content ofthe CW wastewater renders the application of aerobictreatment inecient, mainly due to the cost of oxygen sup-plementation, as well as the generation of higher sludgequantities and odours (Gavala et al., 1999b). In manyAnaerobic digestion of cheese whey wastewaters (CW) was investigated in a system consisting of an ecological pretreatment followedby upow anaerobic lter (UAF). The pretreatment was conducted to solve the inhibition problems during anaerobic treatment of CWcaused by the amounts of fats, proteins and carbohydrates and to avoid the major problems of clogging in the reactor. The optimizedecological pretreatment of diluted CW induce removal yields of 50% of chemical oxygen demand (COD) and 60% of total suspendedsolids (TSS) after acidication by Lactobacillus paracasei at 32 C during 20 h and neutralization with lime. The pretreated CW was usedto feed UAF (35 C). The eects of organic loading rate (OLR) and hydraulic retention time (HRT) on the pretreated CW anaerobicdegradation were examined. The average total COD removals achieved was 8090%. The performance of the reactor was depressedby increasing the COD concentration to 20 g/l (OLR = 4 g COD/l d) and the COD removal eciency was reduced to 72%. Signicantmethane yield (280 l/kg COD removal) was obtained at an HRT of 2 days. 2007 Elsevier Ltd. All rights reserved.
Keywords: Cheese whey; Lactobacillus paracasei; Acidication; Precipitation; Upow anaerobic lter
1. Introduction recovery of the valuable constituents contained in wheyEcological clarication of chdigestion in upo
H. Gannoun, E. Kheli, H. Bou
Laboratory of Microbial Ecology and Technology,
National Institute of Applied Sciences an
Received 9 May 2007; received in revised formAvailable onlin0960-8524/$ - see front matter 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2007.12.037ese whey prior to anaerobicanaerobic lter
lagui, Y. Touhami, M. Hamdi *
partment of Biological and Chemical Engineering,
chnology, B.P. 676, 1080 Tunis, Tunisia
December 2007; accepted 12 December 2007January 2008
Tecliquid euent (Achour et al., 2000), the abattoir wastewa-ter (Mabrouk and Hamdi, 2001; Gannoun et al., 2007),olive mill wastewater (Hamdi, 1996) and fruit and vegeta-ble waste (Bouallagui et al., 2003). Currently, the whey pro-duction in Tunisia is estimated at 35.000 ton/year. Duringthe last few decades, this production has increased veryrapidly with the development of the dairy industry. Anaer-obic digestion is essentially viable method for treating highorganic content wastewater coming from cheese-producingplants (Erguder et al., 2001). However, in spite of wastereduction and energy potential, anaerobic digestion is notwidespread in the dairy industry. This is largely due tothe problem of slow reaction rates which require longhydraulic retention time (HRT) and poor process stability.Malaspina et al. (1995) stated that CW a dicult substrateto treat (especially in highly loaded reactors) due to itshigh organic content and its low bicarbonate alkalinity(50 meq/l). Various types of anaerobic reactors have beenused in laboratories to treat anaerobically CW. Patelet al., 1995 examined the application of upow xed lmanaerobic reactor with high COD in the inuent (70 g/l)and achieved a removal of up to 81%. A laboratory UASBreactor was reported for inuent wastewater concentra-tions between 12 and 60 g COD/l. COD removal rates var-ied between 85% and 99%, at an HRT of 6 days and in anOLR range of from 2 to 7.3 g COD/l d. In addition toanaerobic lters and upow anaerobic sludge blanket(UASB) reactors, hybrid digesters and anaerobic sequenc-ing batch reactors (ASBR) are also employed for treatingdairy euents. An ASBR system was reported to providesoluble COD and BOD5 removal rates of 62% and 75%,respectively, at an HRT of 6 h (Banik and Dague, 1997).High COD removal was obtained up to organic load of19.78 g COD/l d with anaerobic digestion of cheese wheyin two-phase reactor followed by a membrane ltrationsystem. However, the ux decline because the formationand compaction of a cake layer on the membrane surfacecaused by the particulate matter inside the porous mem-brane (Saddoud et al., 2007).
The dilution of cheese whey or the combination of twotreatment methods, biological and chemical, is anotherpossible option for reducing the instability and low e-ciency problems caused by its high organic content. Physi-calchemical treatments allow the partial removal of theorganic load by protein and fat precipitations with dierentchemical compounds such as aluminium sulphate, ferricchloride and ferrous sulphide (Karpati et al., 1995; Ruston,1993). However, since the reagent costs are high and thesoluble COD removal is poor in physicalchemical treat-ment processes, biological processes are usually preferred(Vidal et al., 2000). Therefore, one way of improving theperformance of digesters treating wastewaters with highcontent especially the high-rate anaerobic systems, suchas UASB reactors or upow anaerobic lters is to promotean adequate pretreatment of the substrate.
6106 H. Gannoun et al. / BioresourceThe study was conducted to optimize particulate matterremoval by an ecological clarication and to assess thefeasibility of anaerobic digestion of claried cheese wheyusing an upow anaerobic lter fed with various OLR atdierent COD inlet and HRT.
2.1. Cheese whey clarication by lactic bacteria acidication
and lime addition
The fresh CW was collected from a local TunisianCheese Factory (EL Jedaida, Tunisia) which used tradi-tional technologies for cheese manufacture. It was analyzedand stored at 20 C to avoid the acidication and thechemical composition modication of cheese whey. Lacto-bacillus paracasei ssp. paracasei1, which was isolated previ-ously from fresh CW, was maintained on MRS agar (ManRogosa and Shapman).
Acidication of CW with L. paracasei was carried inErlenmeyer asks with a total volume of 150 ml under sta-tic conditions at 32 C. At the end of the fermentation, thepH was adjusted to neutral value with lime addition whichinduces precipitation of some compounds especially pro-teins and grease. A factorial design experiment (Deshayes,1980) was used to study the eect of three factors (temper-ature of CW fermentation, lime dosage and initial CODconcentration) on the COD and TSS removals eciency.In Table 1, the eight rows correspond to the eight experi-ments that should be carried and the three columns corre-spond to the three factors studied. For each factor high (+)and low () levels were tested. The eect of each factor onresponse (COD and TSS removal) was evaluated by thedetermination of Ci coecients which, are calculated asfollows:
Ci RX i Y i8
where Yi is the experimental value obtained (COD and TSSremoval), and Xi the level of the factor (+ or ) in the iexperiment (18). The chemical composition of cheesewhey before and after ecological pretreatment is shown inTable 2.
2.2. Combined system for CW treatment
Acidication of CW with L. paracasei was carried out in2 l batch bioreactor at 32 C. At the end of the fermentation,the pH was adjusted to the neutral value with lime addition,used as a coagulant. A continuously stirred tank reactor(CSTR) with variable working volume was used to feedthe UAF with a biologically pretreated CW. The suspendedsolids were removed by sedimentation in settler. Mixing wasassured by the continuous rotation of the magnetic stirrer.The anaerobic digestion of the pretreated CW was con-ducted in an UAF reactor consisting of glass column of60 cm in height and 10 cm in diameter. The total volume
hnology 99 (2008) 61056111of the reactor is 2 l. The digester was packed with the Flo-cor (U3L3, porosity 95%, specic surface 230 m2 m3) as a
me dosage after lacticmentation (g/l)
COD removal (%) TSS removal (%)
22 3237 4026 2932 38
7 + +8 + + +
Ci (COD) +5.28 +4.91 +2.22.25
Technology 99 (2008) 61056111 6107Ci (TSS) +4.75 +5.5 +3
Table 2Cheese whey wastewater characteristics before and after ecologicalpretreatment
Parameters Raw CW Pretreated CW
pH 4.46 0.3 7.2 0.25Conductivity (ms/cm) 7.6 0.15 5.6 0.12Table 1Factorial design experiment (k = 3, n = 8)
Temperature of lacticfermentation (C)
COD inlet (g/l) Lifer
25 55 0.6+ 32 5.5 (diluted) 1.8
1 2 + 3 + 4 + + 5 +6 + +
H. Gannoun et al. / Bioresourcesupport for the growth of microorganisms. The inoculumwas obtained from an active biogas digester of fruit and veg-etable waste treatment (Bouallagui et al., 2003). The diges-ter was loaded with pretreated CW and operated at theoptimal mesophilic temperature range (35 1 C) underdierent ORL, obtained by varying the COD inlet and theHRT. The system was fed by a peristaltic pump connectedto a programmable timer.
2.3. Technical analysis
The analysis of the samples taken from the euents ofthe digesters was carried out when steady-state conditionswere established. COD was estimated using the methoddescribed by Knechtel (1978). Total solids (TS), total sus-pended solids (TSS), fats, biological oxygen demand(BOD), total nitrogen, fats and volatile fatty acids (VFA)were determined according to the procedure listed in stan-dards methods for the examination of water and wastewa-ter (American Public Health Association, 1992). Theproteins content were determined according to the methodof Bradford (1976). The biogas produced was collecteddaily in plastic bags at room temperature. The total volumewas later determined with a wet gas meter and time to time
BOD5 (g O2/l) 40 2.55 20 1.87COD (g/l) 60 10 25 5TS (g/l) 59 0.5 42 0.5TSS (g/l) 1.5 0.23 0.8 0.1Proteins (g/kg) 125 2 80 1Fats (%) 0.9 0.5 0.6 0.2the methane content was estimated using an ORSATapparatus.
3. Results and discussion
3.1. Optimisation of treatment of CW by lactic acidication
and lime neutralization
The pretreatment step was based on L. paracasei growthon CW, lactose fermentation into lactic acid and precipita-tion of organic matter after lime addition. Fig. 1 shows thatthe kinetics of L. paracasei growth and the decrease of pHdepend on the initial COD. pH is a crucial indicator of fer-mentation progress and its drops occurs due to the metab-olism of sugars by lactic acid bacteria mainly to lactic acid(Adams, 1990). Hydrophobic and electrostatic interactionsbetween caseins and divalent ions especially Ca2+ is pro-nounced at low pH (Walstra, 1990). Tango and Ghaly
19.25 2728 3137.5 4550 62(1999) demonstrated the positive eect of temperatureand pH on lactic acid production from CW using a Lacto-bacillus strain under batch conditions. The lowest TSS andCOD removal were obtained with experiment 1 while thehighest TSS and COD removal were obtained with experi-ment 8. Indeed, the calculated coecient based on theexperimental design, showed that all factors have a positive
0 6 10 12 14 16 18 20Time (hours)
Fig. 1. Eect of COD concentration on the bacterial growth (OD600): 5 g/l(N), 10 g/l (j), 15 g/l (d); and on the pH evolution: 5 g/l (D), 10 g/l (s)and 15 g/l (h) during lactic acidication of CW by L. paracasei 1 at 32 C.
remained at its neutral value, which is favourable to theactivity of methanogenic bacteria. However, under thehighest OLR, the pH decreased rapidly and the conversionof substrate to biogas was reduced due to the inhibition ofmethanogenic bacteria, caused by VFA accumulation andpH decrease. The maximum COD removal eciency of95% was reached at 15 g/l of COD corresponding to anOLR of 3 g COD/l d (Fig. 2b). When increasing theCOD concentration to 20 g/l (OLR = 4 g COD/l d), theCOD removal eciency was reduced to 63% after 80 days.The UAF performance is highly sensitive to the quality ofthe feed of dairy wastes especially, the COD value. Indeed,the yield and kinetics of the biological reactions involved inanaerobic digestion are strongly dependent upon wastecomposition (Archana et al., 1999). The complete degrada-
600 10 20 30 40 50 60 70 80
0 10 20 30 40 50 60 70 80
0 10 20 30 40 50 60 70Time (days)
Fig. 2. Eect of loading rates by varying the inlet COD on pH variation ofthe euent (h) (a), the COD removal (j) (b), methane productivity ()(c) and methane yield (d) (d) during anaerobic digestion of pretreated CWin UAF (HRT = 5 d).
Teceect on COD and TSS removal. The temperature anddilution factor exhibited positive eect on the kinetics ofacidication by lactic fermentation. The precipitation ofproteins by lime addition is ecient when lactic fermenta-tion given the lowest pH. In fact, the comparison betweenthe runs 2 and 8, and then between the runs 7 and 8 showedthat the addition of lime improved COD and TSS removalsonly when lactic fermentation was carried out with dilutedCW at 32 C. High concentrations of organic material andTSS in the raw CW (5060 g/l and 1.3 g/l, respectively)were reduced by precipitation of proteins and grease afterlactic acidication and lime neutralization (Table 2). Theprotein fraction of the TSS obtained after clarication varybetween 40% and 60%. In the light of these results, theoptimized conditions established for ecient ecologicalpretreatment are: lactic acidication of diluted CW usingL. paracasei at 32 C and neu...