Anaerobic treatment of olive mill wastewater and piggery effluents fermented with Candida tropicalis

Journal of Hazardous Materials 164 (2009) 1398–1405

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Anaerobic treatment of olive mill wastewater and piggery effluents fermented with Candida tropicalis Gregorio Martinez-Garcia a , Anbu Clemensis Johnson a,b,∗ , Robert T. Bachmann a,d , Ceri J. Williams c , Andrea Burgoyne a , Robert G.J. Edyvean a a

Department of Chemical and Process Engineering, University of Sheffield, S1 3JD Sheffield, UK School of Environmental Engineering, Universiti Malaysia Perlis, 02600 Jejawi, Perlis, Malaysia Yorkshire-Forward, Victoria House, Victoria Place, LS11 5AE Leeds, UK d Malaysian Institute of Chemical and Bioengineering Technology, Universiti Kuala Lumpur, 1988 Vendor City, 7800 Taboh Naning, Alor Gajah, Melaka, Malaysia b c

a r t i c l e

i n f o

Article history: Received 5 August 2007 Received in revised form 15 July 2008 Accepted 15 September 2008 Available online 24 September 2008 Keywords: Olive mill wastewater Aerobic fermentation Anaerobic digestion Bio-degradation Candida tropicalis

a b s t r a c t Olive mill wastewater (OMW) contains high concentrations of phenolic compounds that are inhibitory to many microorganisms making it difficult to treat biologically prior to discharge in waterways. The total mono-cyclic phenol reduction in OMW in this study was carried out by aerobic pre-treatment using the yeast Candida tropicalis in a 18 L batch reactor at 30 ◦ C for 12 days followed by anaerobic co-digestion. A COD removal of 62% and a reduction in the total mono-cyclic phenol content by 51% of the mixture was achieved in the aerobic pre-treatment. Pig slurry was added as co-substrate to supplement the low nitrogen levels in the olive mill wastewater. Subsequent anaerobic treatment was carried out in a 20 L fixed-bed reactor at 37 ◦ C and HRT between 11 and 45 days. After a long start-up period, the OLR was increased from 1.25 to 5 kg COD m−3 day−1 during the last 30 days, resulting in subsequent increase in −1 overall COD removal and biogas production, up to maximum values of 85% and 29 Lbiogas L−1 reactor day , respectively. Methane content of the biogas produced from the anaerobic digestion ranged between 65% and 74%. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Olive oil extraction using the traditional press and three-phase decanter methods results in the production of a highly contaminated olive mill wastewater (OMW). The annual production of OMW in the Mediterranean region is estimated to amount to over 107 m3 [1]. Typically, the weight composition of OMW is 83–96% water, 3.5–15% organics, and 0.5–2% mineral salts. The organic fraction is composed of sugars (1–8%), N-compounds (0.5–2.4%), organic acids (0.5–1.5%), fats (0.02–1%) as well as phenols and pectins (1–1.5%) [2]. High chemical oxygen demand (COD) of OMW coupled with its phenol content inhibits the natural organic load degrading capability of the micro-flora in water bodies. Phenolic compounds are considered to be persistent and recalcitrant in the environment [3], toxic to most bacteria and fungi and are used as a slimicide and disinfectant [4]. Their high toxicity, even at low concentrations, has motivated the search and improvement of many treatment techniques such as evaporation [5], physico-

∗ Corresponding author at: Department of Chemical and Process Engineering, University of Sheffield, S1 3JD Sheffield, UK. Tel.: +44 0114 222 7506; fax: +44 0114 222 7501. E-mail address: [email protected] (A.C. Johnson). 0304-3894/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2008.09.055

chemical [6,7], filtration [8] and biological (aerobic/anaerobic) methods [9,10]. Anaerobic digestion is the biological conversion of organic material to a variety of non-biodegradable end products including “biogas” whose main constituents are methane and carbon dioxide. Anaerobic digestion of high-strength organic waste produced by small to medium-sized factories is commonly regarded to be economical and environmentally sound compared to the other methods of treatment for the following reasons: a. Seasonal operation of the olive mills pose no major problem for the anaerobic treatment process as digesters can be restarted after several months [11,12]. b. It eliminates odors caused by the free emission of ammonia and other gases [13]. c. It produces biogas which can be used as a fuel. d. Minimum amount of chemicals required. e. Relatively small amounts of biomass produced which can be used as fertilizer [14,15]. An aerobic polishing step of the anaerobic digester effluent may be required prior to discharge into receiving water bodies or the municipal sewer.

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The physico-chemical methods have the disadvantages of high cost and low efficiency [16]. For example, lime precipitation results in a 40% reduction of the organic matter only alongside production of large quantities of sludge which requires costly disposal [17]. Reverse osmosis has over 90% efficiency in removing organic matter, but with high operating cost and sludge-disposal problems [17]. In order to improve the process efficiency it is necessary to eliminate or reduce the phenolic components in the OMW that are inhibitory to methanogenesis (i.e. mono-cyclic phenols) [18]. Thus, using certain aerobic microorganisms may be an attractive treatment option. Previous studies by Sayadi and Ellouz [19] and Vinciguerra et al. [20] have shown that phenolic degradation is possible by certain bacteria, yeast and fungi. For example, studies have shown that the yeast Candida tropicalis has the ability to degrade phenol (concentration up to 2.5 g L−1 ) by utilizing it as sole carbon and energy source [21]. Ruiz-Ordaz et al. [22] and Martinez-Garcia et al. [23] used C. tropicalis for aerobic pre-treatment prior to the anaerobic digestion. This approach does not require dilution and achieves a significant reduction in the phenol and COD load which enhances the anaerobic degradation process. However, the anaerobic digestion of OMW requires provision of an extra nitrogen source in addition to the nitrogen originally present to satisfy the C:N:P ratio (100:2.5:0.5) [24,25]. The high cost of non-sustainable, inorganic nitrogen sources such as urea and other compounds suggests the use of alternative organic sources of nitrogen such as pig slurry. The use of pig slurry or any animal byproducts for anaerobic digestion requires heat sterilisation at 70 ◦ C for 60 min to reduce most pathogens according to the category 3 of the European regulation EEC 1774/2002 [26]. The objectives of the research were to study the (i) reduction of the phenolic content by means of an aerobic pre-treatment step using C. tropicalis, and (ii) investigate the start-up, biogas production and overall COD reduction of a fixed-bed anaerobic reactor co-digesting pig slurry (viable source of nitrogen) with OMW.

yser (model LASA 20, Germany). Moisture content was determined according to British Standard 1016 [27]. Alkalinity was measured according to the standard procedure given in HMSO 1981. The fraction of methane present in the biogas was evaluated using a Pye Series 104 Chromatograph (PYE UNICAM, England) with a mol sieve and silica gel packed column and argon as a carrier gas at around 90 ◦ C on a kathrometer detector. Lipid content was determined by the Bligh and Dyer method [28]. Volatile fatty acids (VFA) were determined using standard gas chromatography. The column used was an EC-1000, GC capillary column from Alltech Associates Inc. with following specifications: phase: EC-1000, length: 30 m, ID: 0.32 mm, film thickness: 0.25 ␮m. All analyses were carried out in triplicate with a relative standard deviation not exceeding 5%.

2. Materials and methods

2.3. Aerobic pre-treatment

The effluent from a three-phase extraction process was obtained from the olive oil mill Aceites San José (Córdoba, Spain). The cosubstrate (pig slurry) was obtained from Longley Farm (South Yorkshire, UK). The samples were collected fresh and stored at −20 ◦ C. The composition of the raw OMW and the co-substrate pig slurry are summarised in Table 1.

An 18 L L.H. Fermenter 2000 Series (L.H. Fermentation, UK) bioreactor was filled with a mixture of OMW (75%) and pig slurry (25% v/v). The mixture was sterilised in situ by means of a water steam regulator pack at 121 ◦ C for a period of 15 min. After cooling the fermenter was inoculated with 100 mL of C. tropicalis batch culture medium. The air input was sterilised by in-line filtration (Nalgene, PTFE membrane, 0.45 mm pore size, 50 mm diameter) and the flow rate was set to 1 L min−1 . The variations in the following parameters pH, COD and mono-cyclic phenol content were monitored daily for 12 days at a stirring speed of 400 rpm and a temperature of 30 ◦ C. The treated effluent was used as feed for the anaerobic digester.

2.1. Chemical analyses The pH of the samples was measured using HANNA pH meters (Hanna Instruments, Singapore), model HI931300. The COD, total organic carbon (TOC), total Kjeldahl nitrogen, dissolved monocyclic phenol and phosphate content were analyzed using Dr. Lange cuvette tests (LCK 346 and LCK 350, respectively) and UV-vis analTable 1 Composition of raw OMW and pig slurry. Parameter

Unit

OMW

Pig slurry

pH COD TOC TKN Lipids Mono-cyclic phenols PO4 -phosphorous Moisture content Alkalinity

[/] [g L−1 ] [g L−1 ] [g L−1 ] [g g−1 ] [mg L−1 ] [mg L−1 ] [%] [mg CaCO3 L−1 ]

5.04 90.0 29.2 1.05 0.02 700 260 94.83 610

9.25 19.20 5.80 2.49 0.01 90.0 320 98.30 5760

2.2. Aerobic yeast culture preparation and inoculum development The culture of C. tropicalis ATCC 32546 was obtained from the American Type Culture Collection (Virginia, USA) and stored at 4 ◦ C. The yeast was revived by adding 0.3 mL of sterile distilled water. This was mixed well and left to soak for 30 min before being transferred to agar slants (Oxoid, UK). Yeast cultures were grown at 24 ◦ C and transferred to fresh nutrient agar slants every 2–3 weeks. The liquid culture medium [22] consisted of ammonium sulphate (NH4 )2 SO4 , 250 mg L−1 ; potassium dihydrogen orthophosphate (KH2 PO4 ), 125 mg L−1 ; magnesium sulphate 7-hydrate (MgSO4 ·7H2 O), 37.5 mg L−1 ; calcium chloride (CaCl2 ), 3.75 mg L−1 ; yeast extract, 18.75 mg L−1 ; 500 mg of phenol L−1 . The BOD:N:P ratio was fixed to 700:5:1. The medium was sterilised by autoclaving at 121 ◦ C for 20 min. 100 mL of the sample medium was placed in a sterile 250 mL Erlenmeyer flask and inoculated with 3–4 colonies of yeast from the agar slabs. The flask was covered with sterile cotton wool and placed in a Raven incubator (LTE Scientific, UK). The temperature was maintained at 30 ◦ C while shaking was at 150 rpm for a period of 24 h using an orbital shaker SI 50 (Stuart Scientific).

2.4. Anaerobic fixed-bed digestion The anaerobic digestion of the aerobically pre-treated OMW was carried out in a 20 L (18 L working volume) glass bell jar (Quickfit, UK) with a multiport lid to ensure the digester was airtight and the sample ports were sealed with self-sealing rubber suba seals (Fisher Scientific, UK). Air trapped in the digester was purged with nitrogen to ensure anaerobic condition. A commercially available rigid packing design (Floccor, UK) was used, which provided a large surface area for bacterial attachment and good flow within the digester. The Perspex buffer tanks in which the digesters were held was filled with water and maintained at 37 ◦ C using Techne TE-10A filament heater (UK). The water surface of the Perspex buffer tank

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was covered with plastic insulating beads beneath a plastic sheet to retain the heat and reduce evaporation. The digesters were fed using peristaltic pumps (Cole-Palmer, USA, model 7553-75) equipped with 6–600 rpm motor, two Masterflex L/S standard pump heads (model 7013-20) and L/S 13 tubing (Cole-Palmer, UK). Temperature and pH were measured in situ using Hanna Instruments HI 8424 (Singapore) microcomputer pH meter. pH was adjusted when necessary by the addition of NaHCO3 (BDH, UK). Biogas production was measured using the Zeal DM3A 0.25 dm3 gas flow meter. The inoculation mixture for the start-up comprised of 2.5 L activated sludge obtained from a local sewage works (Blackburn Meadows, Sheffield, UK) and 1 L filtered (to separate out solids) OMW. One litre of pig slurry was added to provide an additional, initial nitrogen source to counteract the low levels of nitrogen in the pre-treated OMW. In addition, 10 g of (NH4 )2 SO4 was added and the reactor was topped up with tap water. The initial feed to the digester consisted of 20 g yeast extract (supplement for N), with 100 mL OMW made up to 5 L using tap water (OMW:H2 O ratio of 1:50). Once the biomass had developed and the anaerobic process was under way, the yeast extract was skipped and the OMW:H2 O ratio increased gradually until the digester was running on pure aerobically pre-treated OMW effluent with no dilution. Subsequently, the organic loading rate (OLR) was increased to determine the maximum possible treatment capacity. The organic loading rates (OLRs) used after the acclimatisation period were 0.46, 0.63, 1.25, 1.88, 2.5, 3.75 and 5 kg COD m−3 day−1 . The digester was operated in a semi-continuous mode throughout the experimental period. 2.5. Scanning electron microscopy Samples of the microorganisms found in the anaerobic digester were observed using scanning electron microscopy (SEM). The samples were fixed in Karnovky’s fixative (2% paraformaldehyde, 2.5% glutaraldehyde) in 0.1 M sodium cacodylate buffer for 3 h at 4 ◦ C. Secondary fixation was carried out in 2% osmium tetroxide for 1 h at room temperature. Dehydration was performed through a graded series of ethanol of increasing concentration (75% ethanol for 15 min, 95% ethanol for 15 min, 100% ethanol for 15 min, 100% ethanol for 15 min, 100% ethanol dried over anhydrous copper sulphate for 15 min) all the above steps were carried out at room temperature. The samples were air-dried using hexamethyldisilazane (HMDS) as the transition fluid and then immersed in 100% ethanol: HMDS as a 50:50 mixture for 30 min and then in 100% HMDS for 30 min. The fluid was drained and the samples were allowed to air dry overnight at room temperature. Subsequently, samples were mounted on 12.5 mm diameter stubs, coated with approximately 25 nm of gold in an Edwards S150B sputter coater and examined in a Philips PSEM 501B at an accelerating voltage of 30 kV.

Fig. 1. Aerobic degradation of COD and mono-cyclic phenol compounds in the mixture of OMW and pig slurry with C. tropicalis.

anaerobic digestion since methanogenic bacteria are inhibited by acidic wastewaters. 3.2. Anaerobic digestion of aerobically pre-treated OMW with pig slurry: start-up period In the start-up period (≈4.5 months) of the digester the OLR was maintained between 0.33 and 1.11 kg COD m−3 day−1 for the acclimatisation and growth of the methanogenic bacteria. The COD removal efficiency during the start-up period varied between 62% and 91% (Fig. 2). High COD reduction in conjunction with increasing biogas production provided evidence of anaerobic microbial activity and suggests that the process was viable for the treatment of OMW (Fig. 3). The hydraulic retention time (HRT) in the digester varied between 18 and 36 days during the initial 75 days of operation (Fig. 4). The fluctuations were due to the changes in the feed volume during the acclimatisation period. The digester instability (decrease in COD removal efficiency and biogas production) that followed was caused by an OLR increase from 0.55 to 1.11 kg COD m−3 day−1 on day 83, which was maintained for 6 days. Feeding was therefore stopped for about 23 days and resumed with an OLR of 0.46 kg COD m−3 day−1 on day 112 for 11 days followed by 0.63 kg COD m−3 day−1 on day 123 for 9 days corresponding to a HRT of 90 days due to the decreased volume of feed. The alkalinity in the digester was within the range of 1800–2900 mg CaCO3 L−1 as shown in Fig. 5. A gradual increase in

3. Results 3.1. Aerobic pre-treatment of OMW with pig slurry The results from Fig. 1 show that pre-treatment of the mixture of OMW and pig slurry with C. tropicalis reduced the COD and dissolved mono-cyclic phenol content by 62% (75.6–28.7 g L−1 ) and 51% (541–265 mg L−1 ), respectively. During the aerobic pre-treatment of the mixture of OMW and pig slurry, a temporary marginal drop of the pH was noted on day 4. The pH was therefore adjusted to pH 7 using up to 26 g NaOH L−1 OMW. This was essential to prevent problems during

Fig. 2. The variation in COD removal and OLR with time in the anaerobic digester fed with aerobically pre-treated mixture of OMW and pig slurry.

G. Martinez-Garcia et al. / Journal of Hazardous Materials 164 (2009) 1398–1405

Fig. 3. The change in the production of biogas and methane content produced in anaerobic digester fed with aerobically pre-treated mixture of OMW and pig slurry.

Fig. 4. The variation in hydraulic retention time in the anaerobic digester fed with aerobically pre-treated mixture of OMW and pig slurry.

alkalinity was observed from day 76 until a maximum of 3700 mg CaCO3 L−1 was reached on day 105. Following this, a minor drop in the alkalinity was recorded which was in conjunction with the decrease in COD removal and biogas production. The pH and temperature during the start-up period were found to be pH 7.0 ± 0.1

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Fig. 6. Scanning electron micrograph of rod- and cocci-shaped anaerobic bacteria degrading OMW and pig slurry in a fixed-bed anaerobic digester.

and 36.9 ± 0.2 ◦ C, respectively, providing favourable growth conditions for methanogenic bacteria. Biogas and methane production were low during the initial start-up period, which was attributed to the low methanogenic activity in the digester due to the acclimatisation of the microbes and the low OLR used. After 2.5 months of acclimatisation, a substantial increase in the production of biogas alongside higher OLRs −1 on was observed, which reached a value of 7 Lbiogas L−1 reactor day day 79 as shown in Fig. 3. Progressive increase in biogas production was observed during the recovery period. Microbial growth shown in Fig. 6 is evidenced by the presence of rod and cocci-shaped bacteria, which suggest mixed population of bacteria was involved in the biodegradation of OMW. The presence of mixed population is not surprising because in a one-stage anaerobic digester both acidogenic and methanogenic bacteria are present. The micrograph also proves that bacteria were attached to the packing medium which is essential to retain the slow-growing biomass. This is important for treatment at higher OLR to prevent washout of biomass from the anaerobic digester. The overall startup time required for this system was 132 days including a 57 days recovery period from digester instability. 3.3. Anaerobic digestion of aerobically pre-treated OMW with pig slurry: operation The feed used in the operation phase of the digester continued to be aerobically pre-treated OMW and pig slurry. The temperature of the digester was maintained at 37 ± 0.1 ◦ C, and the pH varied between 6.9 and 7.5 (Fig. 7). A gradual increase in the pH was recorded in the last 20 days of the experiment and reached a peak value of 7.8. This increase in pH may be attributed to the high OLR values tested during the final stages of the experiment. 3.3.1. Alkalinity The alkalinity gradually increased from day 133 (3740 mg CaCO3 L−1 ) attaining a maximum of 5120 mg CaCO3 L−1 at the end of the experiment. This high alkalinity value indicated that the system produced enough alkalinity during the anaerobic digestion process.

Fig. 5. The variation in alkalinity in the anaerobic digester fed with aerobically pretreated mixture of OMW and pig slurry.

3.3.2. Increase in OLR On day 133 the OLR was raised from 0.63 to 1.25 kg COD m−3 day−1 . The reactor responded well with COD removal efficiencies >75%. Due to time constraints, it was decided to step up the OLR instead of using gradual increments. The incre-

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4. Discussion 4.1. Aerobic pre-treatment of OMW with pig slurry

Fig. 7. The variation in pH and temperature in the anaerobic digester co-digesting aerobically pre-treated OMW with pig slurry.

ments were made on day 139 with an OLR of 1.88 kg COD m−3 day−1 , day 145 with OLR 2.50 kg COD m−3 day−1 , and day 153 with OLR 3.75 kg COD m−3 day−1 . At the final days of the experiment (days 157–161), the anaerobic digester was operating at an OLR of 5 kg COD m−3 day−1 . The increase in OLR corresponds to a HRT decrease from 45 to 11 days. The COD removal efficiency varied between 78% and 91% with OLRs greater than 1 kg COD m−3 day−1 .

3.3.3. Volatile fatty acids VFAs were determined for the sample taken on the last day before the digester was shut down. Acetic acid (59.5 mg L−1 ) and propionic acid (61.3 mg L−1 ) were observed as the main metabolic products of the acidogenic stage.

As seen from Fig. 1, C. tropicalis was capable of reducing the COD by 62% and the total mono-cyclic phenol by 51%. Similar results have been reported by Martinez-Garcia et al. [23] using C. tropicalis for the co-digestion of OMW and cheese whey mixture. Gharsallah et al. [29] studied the aerobic fermentation of OMW using the white rot fungus Phanerochaete chrysosporium and reported a COD reduction of 49%. Higher phenol reductions were observed with other microorganisms. Yesilada et al. [30] reported that the biodegradation of OMW with the white rot fungus Funalia trogii resulted in a 72% removal of phenolic compounds and 40% COD. In another work by Yesilada et al. [31] with the white rot fungi Trametes versicolor, a reduction of phenolic compounds by 88% and COD by 50% was observed. Chtourou et al. [32] reported that greater COD and phenol reductions (>80%) can be achieved by using the yeast Trichosporon cutaneum for OMW with an initial COD of 19–72 g L−1 . From studies on activated sludge digestibility it is known that concentration of soluble COD and speed of hydrolysis increased when subjected to sterilisation prior to biological treatment [33,34]. Fountoulakis et al. [35] investigated the effects of OMW sterilisation, thermal processing and dilution on phenol biodegradation by the white rot fungus Pleurotus ostreatus. The authors observed that aerobic phenol degradation was greatest in sterilised, 50% diluted OMW (78.3%) after a 21-day incubation period. It was also noted that the mycelium yield was significantly lower in sterilised than in thermally processed OMW indicating that chemical composition and nutritional value are less affected in the latter. The observations from both reports suggest that the autoclaving step deployed in this study was beneficial for the aerobic breakdown of phenolic compounds and possibly overall COD reduction. 4.2. Start-up period of anaerobic fixed-bed digester

3.3.4. Biogas After 134 days of operation, the biogas production began to increase rapidly with increase in the OLR. Peak biogas pro−1 was achieved with an OLR of duction of 29 Lbiogas L−1 reactor day −3 −1 5 kg COD m day . The biogas production was plotted as a function of OLR (Fig. 8) proving the direct linear correlation between the biogas production and the OLR. With increasing OLR the methane content of the biogas decreased slightly. Nevertheless, methane yields of 65–74% were obtained throughout the entire process.

Fig. 8. The variation in biogas and methane produced during anaerobic co-digestion of OMW with pig slurry in a fixed-bed reactor at various OLR.

Initial start-up period (≈4.5 months) of the anaerobic digester was sufficient to enrich the system with biomass and acclimatise the microbes to OMW and aromatic compounds present. During this period, the COD reduction reached a peak of 91% with COD of the influent ranging between 20 and 56 g L−1 . Longer overall start-up times as experienced in the present study may be reduced considerably if lab- or industrial-scale digesters are inoculated with large amounts of acclimatised biomass. During the start-up period of the anaerobic digester acidogenic bacteria use compounds which can be easily metabolized, such as sugars and carbohydrates, whereas methanogens which have a slower metabolism are hindered by the presence of aromatic compounds and volatile acids [36] resulting in lower biogas production as also observed in this study. Insufficient alkalinity during the start-up period resulted in variations in pH, which in turn negatively affected the performance of the acidogenic/methanogenic consortium as evidenced by a decrease in COD removal. Severe pH changes are often caused by alterations in the organic or hydraulic parameters, temperature and presence of toxic substances. Toxic substances inhibit anaerobic digestion by altering the kinetics of organic degradation. These factors can alter the methanogenic activity vital for the anaerobic digestion process. As a consequence, an imbalance in the simultaneous process of acidogenesis and methanogenesis can lead to the acidification of the system. This can eventually inhibit the production of biogas [10]. In this study, the decline in pH is thought to be due to an accumulation of volatile acids caused by an imbalance between volatile acid production and conversion to biogas during the start-up period of

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the anaerobic digester and the presence of residual phenolic compounds after aerobic pre-treatment. In addition, the pH decrease may also be attributed to the semi-continuous feeding mode. Semicontinuous feeding is more convenient at laboratory scale, does not require sophisticated and expensive process control technology and does not compromise the quality of study of the anaerobic digestion process [37]. On the other hand, batch feeding creates a cycle of feeding-starvation. Because of the faster metabolism of the acid and hydrogen-producing bacteria compared with the acid and hydrogen-consuming bacteria, there is a sudden increase in acid and hydrogen production in the digester. This may lead to a fall in pH if the alkalinity in the system is not sufficient to neutralize the surge of organic acids. Moreover, these surges in acid may produce high concentrations of ammonia–nitrogen. The presence of ammonia–nitrogen is beneficial at concentrations between 50 and 200 mg L−1 as an essential nutrient; however it may be detrimental at high concentrations [38,39]. In order to prevent rapid pH changes and inhibition of methanogenic bacteria, the digester must have an adequate buffering capacity. Sodium bicarbonate was added to increase the alkalinity in the digester during the periods of pH declination. Excessive addition of alkalinity was avoided to prevent ammonia toxicity and precipitation leading to sludge formation. pH was found to increase during the recovery period, which is an indicator of the good performance of the digester. 4.3. Operation of the anaerobic fixed-bed digester Throughout the entire period of study the anaerobic fixed-bed digester was mostly stable and recovered from overloads in COD causing pH fluctuations. The pH remained within the range of pH 6.9–7.8 throughout the experiment once conditions had stabilized. This pH stability guards the digester from possible acidification and provides an optimum environmental condition for methanogenic bacteria. The maximum OLR tested was 5 kg COD m−3 day−1 and an average COD removal efficiency of 85% was achieved in the anaerobic digestion with OLR > 1 kg COD m−3 day−1 . Comparison of the data from this study with other anaerobic studies using OMW showed that the COD removal efficiencies vary considerably according to the type of digester and the microorganisms used in the pretreatment (Table 2). Fixed-bed studies obtained efficiencies in terms of COD removal varying between 55% and 83%. However, these digesters did not cope very well with high OLRs, with a maximum of up to 8 g COD L−1 day−1 . The results obtained in this study are among the best, only surpassed by those of Rigoni-Stern et al. [40] who found a COD removal of 87%. The results from this study suggest that a two-stage process can be successful provided the

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OMW and co-substrates are available in the required amounts and within a reasonable distance to avoid incurring excessive transportation costs. Co-digestion of OMW with pig slurry as carried out in this study is suggested to increase the degradability of OMW. Other researchers who carried out studies on the co-digestion of OMW with other wastes corroborate this suggestion. Marques et al. [41] reported the anaerobic digestion of undiluted OMW mixed with piggery effluents. In their study, the OMW was treated successfully, increasing the influent COD from 25 to 66 kg COD m−3 at a HRT of 6–7 days and achieving total COD removal between 70% and 83%. However, aerobic pre-treatment was not carried out and OMW was mixed with 50% piggery effluent, which decreased the influent COD and reduced the concentration of polyphenols in the solution, thus easing the degradation process. The efficiency of the anaerobic purification system decreases when the concentration of the OMW with respect to the co-substrate increases, since the mixture becomes harder to degrade. Marques et al. [42] observed that the process was stable in a volumetric proportion of 17–83% (v/v). However, when the concentration of the OMW reached 91%, an imbalance in the process was observed, which was attributed to the lack of ammonium–nitrogen. In this study, COD reduction during anaerobic co-digestion of OMW with pig slurry varied between 62% and 89% at OLRs of 0.20–0.44 kg COD m−3 day−1 . Similar observations were made by Gharsallah et al. [29]. COD removal efficiency in excess of 75% could not be achieved at the lowest loading rates (0.9 kg COD m−3 day−1 ). On the other hand, Boubaker and Ridha [43] have shown that a maximum COD removal of ∼90% could be achieved by increasing the HRT to 36 days at lower OLR (0.67 kg COD m−3 day−1 ). This suggests that high molecular weight aromatics are not degraded anaerobically within a few days. On the other hand, the organics degraded by anaerobic microorganisms were mainly transformed to methane. Thus, the aforementioned results can be compared with the present study and it could be suggested that the inhibition observed at certain periods during the experiments might be due to the presence of polyphenols that although reduced to a greater extent, still posed a problem. Further evidence for the inhibitory effects of polyphenols was provided by Sayadi et al. [44] who showed that the high molecular mass fraction of polyphenols in high-strength OMW (COD > 55 g L−1 ) inhibited the production of lignin peroxidase in the white rot fungi P. chrysosporium and caused a decrease in anaerobic polyphenol degradation and COD removal, respectively. Beccari et al. [45] reported that in a two-stage anaerobic digestion process (acidogenic–methanogenic) of OMW no degradation of polyphenol-like substances was observed under acidogenic conditions whereas some polyphenols were degraded in the methanogenic stage. Sorlini et al. [46] studied the polyphenols

Table 2 Performance comparison of various two-stage processes using OMW as feed. Culture

Aspergillus terreus Aspergillus niger Geotrichum candidum Azotobacter chroococcum Aspergillus terreus Phanerochaete chrysosporium Geotrichum sp. Aspergillus sp. Candida tropicalis Candida tropicalis Candida tropicalis

Aerobic pre-treatment

Anaerobic digestion

References

COD removal (%)

Phenol removal (%)

Loading rate (g COD L−1 day−1 )

COD removal (%)

57.8 61 63.3 74.5 74 51 55 52.5 62.8 62 62

94.3 58 65.6 90 94.3 – 46.6 44.3 51.7 54 51

4.4 10 (batch)

70 55

– –

[50] [51]

2.3–3.4

–

–

[52]

1.3–2.4

75

75

[29]

–

–

[53]

83 85

75 76

6–24 (batch) 3 5

Max. methane content (%)

[23] This study

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Table 3 Volatile acids present in the effluent from the anaerobic digester before shut down. Compounds

Amount present (mg L−1 )

Ethanol Acetic acid Propionic acid iso-Butyric acid Butyric acid iso-Valeric acid Valeric acid Caprionic acid

5.02 59.45 61.32 7.78 7.66 7.25 7.09 6.41

in OMW and identified them as being responsible for inhibition of the anaerobic process. Of the three groups of bacteria (hydrolytic, acetogenic and methanogenic) involved in the anaerobic digestion, hydrolytic bacteria were not sensitive to polyphenols, acetogenic bacteria were sensitive to caffeic acid at concentrations >0.25 g L−1 and methanogenic bacteria were sensitive to caffeic, ferulic and cumaric acids at concentrations exceeding 0.12 g L−1 . Borja et al. [47] observed that phenols which were degradable (tyrosol and p-hydroxybenzoic acid) stimulated methane production. Another study carried out by Kouroutzidou et al. [48] demonstrated that anaerobic microorganisms were able to decompose gallic acid at concentrations as high as 1000 mg L−1 . Martinez-Garcia et al. [23] reported that the inhibition of the anaerobic process was caused by the build up of VFA (acetic acid and propionic acid). Although the presence of VFA was not monitored regularly in this study, acetic and propionic acid predominated in the effluent from the anaerobic digester (Table 3). However, the concentration of VFAs observed in this study were low and indicative of good process stability and performance as reviewed elsewhere [49]. Boubaker and Ridha [43] observed that short HRTs (12 days) can reduce the COD removal efficiency and the methane content of the biogas of the anaerobic digester. In this study, the COD removal efficiency of the anaerobic digester at the final days of operation appeared to decrease slightly from a peak of 91–85%. However, conclusive inferences cannot be made owing to the relatively short duration of operation at higher OLRs. Longer HRTs results in the retention of non-attached anaerobic biomass in the digester for increased degradation of organic compounds and to keep the anaerobic process stable. Short HRTs, on the other hand, can lead to wash out of the suspended fraction of anaerobic microorganisms from the digester resulting in reduction in COD removal. 5. Conclusion Initial aerobic pre-treatment of autoclaved OMW was carried out in an 18 L batch reactor at 30 ◦ C for 12 days using the yeast C. tropicalis. This was followed by anaerobic co-digestion with piggery effluent in a 20 L fixed-bed reactor at 37 ◦ C and HRTs between 11 and 45 days. Piggery effluent was used as a co-substrate to compensate for low nitrogen content in OMW. The results of this study illustrate that aerobic pre-treatment combined with anaerobic co-digestion not only reduces monocyclic phenolic compounds by 51%, but also reduces the overall COD content in the effluent by up to 85%. Biogas production during anaerobic digestion reached a maximum value of 29 −1 at an OLR of 5 kg COD m−3 day−1 , and the Lbiogas L−1 reactor day methane content of the biogas varied between 65% and 74%. Despite overall COD removal efficiencies in the order of 85%, aerobic post-treatment should follow to further decrease the COD of anaerobically digested OMW prior to discharge.

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