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BACKGROUND: Sal (Shorea robusta) deoiled seed cake extract (SDOCE) was assessed for its suitability as a cheaper natural substrate for lipase production under submerged fermentation for the first time. The bacterial isolate Aeromonas sp. S1 isolated from dairy wastewater sludge was used for lipase production. Both the isolate and its lipase were also shown as potential bioremediation tool for treatment of dairy wastewater containing higher organic load.
RESULTS: On substituting tributyrin with SDOCE, the lipase production could be enhanced to 24-fold (195 Uml-1) compared to initial 8.13 Uml-1 of lipase activity. Maximum lipase production was obtained at pH 8.0 and incubation temperature of 30°C. The lipase had pH and temperature optima of 10.0 and 55°C respectively. The isolate and its crude enzyme preparation were checked separately for applicability in dairy wastewater treatment. The isolate was able to reduce 93% of COD, 75% of O&G and 47% of TSS after 96 h of treatment. Whereas, enzymatic preparation showed 86% reduction of COD after 12 h and 75 and 45% reduction of O&G and TSS respectively after 96 h of treatment.
CONCLUSION: Over all the study shows the usefulness of Sal seed deoiled cake, a cheaper agro-industrial by-product for the production of lipase. The isolate and its lipase can also be effectively used for bioremediation of dairy wastewater.
Key words: Sal seed cake; Aeromonas sp; Lipase; Dairy wastewater; Bioremediation
Lipases (triacylglycerol acylhydrolases, E.C. 18.104.22.168) are enzymes that catalyze the hydrolysis of triacylglycerols to glycerol and free fatty acids at the water-lipid interface and the reverse reaction in non-aqueous media.1 Due to their multifaceted properties which find usage in a wide array of industrial applications, such as food technology, detergent, chemical industry and biomedical sciences, lipases have emerged as key enzymes in swiftly growing biotechnology.2 Bioremediation for waste disposal is a new avenue in lipase biotechnology3. Lipase have been successfully used for hydrolysis/treatment of high lipid content wastewater originating from dairy and other food processing industries.4,5
Due to extensive industrial and environmental applications of lipases, efforts have been directed to explore the means to reduce the lipase production costs through improving the yield. The use of either cost-free or low-cost agricultural byproducts as raw material for lipase production can be one step to lower the overall enzyme cost.6 In the recent past, many agro-industrial byproducts such as wheat bran, rice bran, molasses bran, barley bran, maize meal, soybean meal, potato peel, coconut oil cake, etc. have been screened as low-cost solid substrates for microbial lipase production.6, 7 There are several reports describing production of various enzymes using oil cakes as substrate in solid-state fermentation (SSF), or as supplement to the production medium.8 Oil cakes/oil meals, by-products obtained after oil extraction from the seeds are ideally suited nutrient support rendering both carbon and nitrogen sources, and reported to be good substrate for enzyme production using microbial species.8
Shroea robusta, commonly called as Sal, is one of the important forest tree, abundantly found in central and parts of eastern and northern India. Sal seeds are an important forest byproduct available from these forests. The Sal seeds are rich in seed fat, which contains about 69 per cent symmetrical triglycerides, makes its potential for the food sector.9 It forms the primary ingredient for a diverse range of products such as oil, soap, cocoabutter equivalent (CBE) in chocolate manufacturing and is also used for tanning purposes.9 Sal seed is now potentially identified as one of the major source of biodiseal production.
At present about 1.50 million metric tones of Sal seeds are produced per year in India, which generates around 1.32 million metric tones of deoiled cake after oil extraction.10 At present it is generally used for non biological processes such as fuel for boiling in solvent extraction plants, as a sizing material in textile industries, coal briquettes and as cattle feed after standardization, as it is available at cheaper cost of Rs. 4/- per kg.9 Sal seed cake contains approximately 7.3% of moisture, 94% of dry matter, 11.7% of crude protein, 2.7% of lipid 13.5% of crude fiber, 5% of ash, 43% of starch, 5% of reducing sugars and 9% of tannins.11 This composition is appropriate for various bioprocesses and its meaningful utilization.
There is no report on using Sal seed cake for enzyme production to best of our knowledge. The present study aimed at developing appropriate bioprocess for enhanced lipase production from isolate Aeromonas sp. S1, using cheaply available Sal seed cake. And potential application of the isolate and its lipase for treatment/hydrolysis of dairy wastewater.
The media components were purchased from Hi Media Laboratories (Mumbai, India). The p-nitrophenyl palmitate (pNPP), substrate for lipase assay was obtained from Sigma Chemical Co. USA. All other chemicals used were of analytical grade. Sal (Shorea robusta) deoiled seed cake was obtained from local Sal oil extraction unit in Raipur, Chattisgarh, India and Mahua (Madhuca sp. ) flowers were collected from local people of Jharkhand, India.
Screening, isolation and purification of microbes
For isolation of lipase producing microbes, the samples were collected from local dairy industry, New Delhi, India. These samples were taken from sludge of oil and grease chamber, and soil enriched with dairy wastewater from proximity of dairy wastewater treatment plant. The samples (1 g) were suspended in 10 ml of sterilized distilled water and the resultant suspension was spread on nutrient agar plate. The plates were incubated for 24 h at 30Â°C. Growing colonies were further purified by repeated streaking on nutrient agar plates. A total of four different isolates could successfully be isolated from sludge samples while four different isolates were purified from soil sample.
Selection of lipase producers among isolated microbes
All the eight isolates were checked for lipase production. The isolates were first spotted individually on tributyrin agar plates (pH 7.5), containing (gl-1): Peptone, 5; NaCl, 5; Yeast extract, 1.5; Beef extract, 1.5; agar, 15 and Tributyrin, 1% (v/v). Production of clear halo zones around the colonies confirmed the lipase production by the isolates. The four isolates (S1, S6, S7 and S8) which showed positive test on tributyrin plates were then checked for lipase production in liquid media.
Media and Culture conditions for lipase production
Mother cultures were prepared by inoculating a loopful of stock cultures of all the four selected isolates (S1, S6, S7, S8) individually in nutrient medium (pH 7.5) containing (gl-1): Peptone, 5; NaCl, 5; Yeast extract, 1.50; Beef extract, 1.50 followed by incubation at 30Â Â°C and 200Â rpm in an orbital shaker. Three hundred microlitre of the above overnight grown mother cultures were used to inoculate 100 ml of above medium containing 1% (v/v) tributyrin (pH 7.5) in 250 ml Erlenmeyer flasks individually. The flasks were incubated at 30Â°C with constant shaking at 200 rpm in an orbital shaker (Brunswick, USA). Aliquots of various samples were withdrawn at different time intervals and centrifuged at 12000 rpm and 4°C for 10 min to pellet down the cell mass. The supernatants thus obtained were used for lipase assay.
All the four isolates were found to produce extra cellular lipase in liquid media. The best lipase producing isolate (S1) was selected for further use. S1 was originally isolated from sludge sample collected from oil and grease chamber of dairy wastewater treatment plant. Isolate S1 was maintained on nutrient agar slants at 4Â°C and subcultured every 20 days interval for further studies.
Identification of lipase producing strain S1
Isolated and purified potential lipase producing bacterium (isolate S1) was identified to be Aeromonas sp. from Microbial Type Culture Collection Facility (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India and deposited at their National Facility with Accession No. MTCC 10661.
Effect of various natural substrates on lipase production
Carbon sources in the form substrate are known as an important factor affecting yield and cost of enzyme production. Carbon substrate, tributyrin, was replaced with cheaper natural substrates like Sal seed deoiled cake extract (SDOCE) and Mahua flower extract (MFE) to see their effects on lipase production by Aeromonas sp. S1.
Preparation of Sal deoiled cake extract (SDOCE) and Mahua flowers extract (MFE)
To 10 gm of grounded deoiled Sal seed cake and Mahua flowers, 100 ml of distilled water was added to make individual slurries. After boiling for 10 min, the slurries were filtered through muslin cloth and filtrates thus obtained were used as substrate's extracts. Three percent of each extract was added to above nutrient media; in case of studying the effects of different nutrient supplements on lipase production. Maximum lipase production was obtained with SDOCE after 24 h of fermentation henceforth this time period and nutrient media containing SDOCE was used for further optimization studies.
Effect of culture conditions on lipase production
Fermentation was carried out in nutrient media containing SDOCE as supplement nutrient source or substrate to study the effects of various nutritional and physical factors on lipase production by Aeromonas sp. S1. Different concentration of SDOCE extract (1%, 3%, 5%, 10%; v/v), Initial pH of the medium (5, 7, 8, 9 and 10), incubation temperature ( 15 °C, 25 °C, 30 °C, 35 °C and 40 °C), carbon sources (Mustard oil, 1% (v/v); Coconut oil, 1% (v/v); maltose, 1% (w/v) and dextrose, 1% (w/v) and nitrogen sources (sodium nitrate, 1%; ammonium chloride, 1% and ammonium sulphate, 1% (w/v) were the parameters used for optimization studies. All the conditions were varied, one at time, in the above media and their individual effect was monitored on lipase production. After growth, cultures were harvested by centrifugation at 12000 rpm and 4°C for 10 min at 4°C. Lipase activity was determined in cell free supernatants. Unless otherwise mentioned, lipase production was carried out by inoculating nutrient media with 3% (v/v) of SDOCE extract (pH adjusted to 8.0) with 300 Âµl of Aeromonas sp. S1 mother culture followed by incubation at 30 °C and 200 rpm. After 24 h of growth, the samples were centrifuged and the supernatant thus obtained was used as crude enzyme preparation.
Lipase activity in the cell free supernatant was determined as described by Mahanta et al.12, using pNPP as the substrate. Briefly, 200Â Î¼l of cell free culture supernatant (crude lipase preparation) was added to 1.8 ml of solution containing 0.15Â M NaCl and 0.5% Triton X-100 in 0.1Â M Tris-HCl buffer (pH 8.0). Twenty microliters of substrate (50Â mM pNPP in acetonitrile) was added to the reaction mixture and incubated at 37Â Â°C for 30Â min. The amount of liberated p-nitrophenol (pNP) was recorded at 400Â nm (UV-Visible spectrophotometer, HACH). One unit is defined as the amount of enzyme liberating 1Â nmol of pNP under standard assay conditions.
Effect of pH on crude lipase was studied by assaying the enzyme at different pH values in the range of pH 4.0-11.0, using pNPP as the substrate.
To determine the optimum temperature, the activity of lipase was measured at various temperatures (25 - 75°C) using pNPP as the substrate. The thermal stability was studied by incubating the enzyme at 55Â°C. Aliquots were withdrawn at different time intervals and the residual activities determined at assay temperature.
Preliminary treatment/hydrolysis of Dairy waste water
Collection and characterization of dairy wastewater sample
Fresh inlet dairy wastewater (raw) was collected from local dairy industry located at New Delhi, India. The wastewater sample was characterized for the following parameters; pH, temperature, COD (Chemical Oxygen Demand), BOD (Biological Oxygen Demand), TSS (Total Suspended Solids), O&G (Oil & grease), total nitrogen, total phosphorus and total coliforms. The pH and temperature of the sample was analyzed on the spot. For BOD estimation, wastewater samples were collected in BOD bottles (non-reactive borosilicate glass bottles of 300 ml capacity). BOD and COD were determined on the same day, after bringing the sample to the laboratory. Whereas, analysis of other physico-chemical and biological parameters was started as soon as possible after collection to avoid unpredictable changes in water sample. The O&G content was determined using partition gravimetric method.13 Other parameters were estimated according to standard APHA method.14
The rest of the sample was stored in cold room (4Â°C) for further studies.
For the treatment of dairy wastewater samples, microbial sample was prepared by inoculating the loopful of stock-culture of Aeromonas sp. S1 in 250 ml Erlenmeyer flask containing 100 ml of nutrient media. The flask was then incubated overnight at 30Â°C and 200 rpm in an orbital shaker.
For the treatment, 5% (v/v) of this overnight grown microbial culture was added in a two liter Erlenmeyer flask containing 500 ml of raw waste water. Before inoculation, pH of raw waste water was adjusted to 9.0, using 0.1N NaOH. After inoculation, the flask was incubated at 37Â°C and 200 rpm for 96 h.
For the enzymatic treatment of dairy wastewater, Aeromonas sp. S1 lipase was used. Production of lipase was carried out under optimized conditions as mentioned in section 2.7: nutrient media containing, 3% (v/v) SDOCE; initial pH, 8.0; inoculum size, 300Âµl and incubation temperature, 30Â°C. The sample was withdrawn after 24 h of fermentation and centrifuged at 12000 rpm, and 4Â°C for 10 min. This cell free supernatant was used as crude enzymatic preparation for dairy wastewater treatment.
pH of raw waste water was adjusted to 9.0, using 0.1N NaOH before treatment. Five percent of above mentioned crude lipase preparation (cell free) was added to 500 ml of raw waste water (in a two liter Erlenmeyer flask). The treatment was carried out at 37Â°C and 200 rpm for four days.
During treatment, all the samples were withdrawn periodically including zero day samples for estimation of COD, O&G and TSS changes. A control flask without microbial (Aeromonas sp. S1) inoculation/ enzyme preparation was also kept under similar conditions.
Each experiment in case of effect of culture conditions and enzymatic characteristics were done at least two times and the differences in their individual results in each set of experiments were less than 5%.
RESULTS AND DISCUSSION
The primary objective of the work was to use the SDOCE as a substitute of tributyrin and inducer/carbon source for cost-effective production of lipase under submerged fermentation.
The other major objective was to find out the possible potential application of this lipase in bioremediation of wastewater originating from food processing industries. Since the utilization of commercial enzymes for treatment/prehydrolysis is expensive along with requirement of high enzyme yield for treatment.15 Thus utilization of cheaper and easily available SDOCE as natural inducer for enzyme production can reduce the cost of enzyme production and hence the treatment cost.
Isolation, purification and identification of potential lipase producing microbes
Total of eight different isolates (S1-S8) were isolated from samples collected from local dairy industry. During screening for lipase production, four isolates (S1, S6, S7 and S8) showed positive test on tributyrin plates.
These four isolates (S1, S6, S7 and S8) were then checked for lipase production in nutrient media containing 1 % (v/v) tributyrin as inducer. Fig. 1 shows the time course production of lipase by different isolates in nutrient media containing 1% (v/v) tributyrin. The lipase activity was found to be maximum (16.8 Uml-1) by isolate S1, on third day of fermentation. Thereafter, the enzyme production started decreasing. While the second highest enzyme producing isolate was found to be S6, showing 4.2 Uml-1 of enzymatic activity on third day of fermentation. Isolates S7 and S8 were found to show minimum lipase activity of less than 4 Uml-1.
The isolate S1 showing maximum lipase production was selected as the potential isolate for further studies. Isolate S1 was identified morphologically (based on physiological and biochemical characteristics) as Aeromonas sp. from Microbial Type Culture Collection Facility (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India and deposited at their national culture collection facility with accession number MTCC 10661.
Effects of various natural substrates on lipase production
In a recent review paper, Treichel et al.1 have reported that the combination of both synthetic mediun (defined compounds) and agroindustrial residues can be used for the purpose of lipase production. In view of this, the combination of nutrient media with agroindustrial/forest by-products for enhanced lipase production was attempted in the present study also. The time course of lipase production by Aeromonas sp. S1 in the presence of different substrates is presented in Fig. 2(a). The results show that lipase activity was found to be maximum in case of SDOCE as supplement substrate. The enzyme production was highest (107 Uml-1) at 24 h of fermentation and decreased thereafter. MFE was also found to produce good enzyme production in 24 h (27 Uml-1), but less compared to SDOCE. In case of SDOCE and MFE, the lipase production was enhanced to approx. 13-fold (107 Uml-1) and 3-fold (27 Uml-1) respectively compared to initial 8.13 Uml-1 in presence of tributyrin at 24 h. This shows that cheaper SDOCE could efficiently substitute tributyrin as supplement substrate to work as inducer and carbon source for enhanced and cost-effective lipase production.Â
There was minimal production of lipase (13.8 Uml-1 in 72 h), in media without any additional substrates i.e. nutrient media only. However addition of SDOCE increased the production to 107 Uml-1 in 24 h of fermentation. This shows that lipase can be produced more and early also, with supplementation natural substrates. Control tests were also done, in which enzyme preparations were first boiled, at 100 °C, for 10 min and then usual assay was performed using pNPP as substrate. No lipase activity was detected in the control sets (data not shown). Since maximum lipase production was observed in case of SDOCE after 24 h of fermentation, henceforth nutrient media with 3% (v/v) of SDOCE as basal media and incubation time of 24 h was used for further optimization studies. Lotrakul and Dharmsthiti16 have reported that addition of soybean meal (by product of vegetable oil industry) and whey (a dairy industrial waste) to a defined media increased 10-fold lipase (450 Uml-1) production to that in nutrient media (40 Uml-1), using Aeromonas sorbia LP004 as lipase producer. Similarly Rajagopalan and Krishnan 17 have shown that addition of sugarcane bagasse hydrolysate SBH (1% reducing sugar (w/v)) to the nutrient medium supported maximum a--amylase production of 67.4 Uml-1 by Bacillus subtilis under submerged fermentation.
Since maximum lipase production was observed in case of SDOCE, among various substrates tested, the effect of concentration of SDOCE on lipase production was looked into. Fig. 2(b) show that SDOCE enhanced lipase production with an optimum at 3% (v/v) SDOCE concentration beyond this led to slight decrease in lipase production.
According to Gupta et al.2, lipase are considered as inducible enzymes thus their production is strongly influenced by carbon sources such as triglycerides, sugars, sugar alcohol, polysaccharides, whey, casamino acids and other complex sources. SDOCE does not contain significant amount of residual oil but has major amount of total carbohydrates in the form of starch polysaccharides11, which might have played an important role in enhanced lipase production by Aeromonas sp. S1. Immanuel et al.18 and Gunashekaran et al.19 have also reported enhanced lipase production in presence of starch using Serratia rubidaea and Citrobacter freundii IIT-BT L139 respectively.
Effect of pH on lipase production
To check the influence of pH on lipase production in the presence of SDOCE, fermentation was carried out at varying initial pH of the medium. The lipase production was found to be greatly affected by pH variation as shown in Fig. 3(a). Maximum enzyme activity was observed at pH 8.0. Very poor enzyme activities were observed at pH 5.0 (5%), 7.0 (32%) and 10.0 (20%), while at pH 9.0 good lipase production of 57% was detected. Similar pH optima of 8.0 for maximum lipase production have also been reported by Immanuel et al.18 and Singh et al.20 in case of Serratia rubidaea and Pseudomonas aeruginosa respectively.
Changes in pH of fermentation media by Aeromonas sp. S1
The pH of fermentation media may change during fermentation due to production or utilization of acidic or alkaline compounds.12 The Samples from fermented media with varying initial pH values were aseptically withdrawn after 24 h of growth and pH determined. As shown in Fig. 3(a), the pH of both low (acidic) and high value (alkaline) were changed to neutral to slightly alkaline range (6.6 - 8.6) after 24 h of growth at 30°C. Increase in pH from initial value of 5 to final value of 8.0 is also reported by Gombert et al.21 during lipase production by Penicillium restrictum in solid state fermentation using Babassu cake. In submerged fermentation also the similar change in pH was reported by Freire et al.22 Because, in this study, the pH for maximum enzyme production was also found to be 8.0 - 9.0, this supported the findings. This property of changing pH is very interesting, and has useful applications in the treatment of wastewater. This will reduce the cost of treatment, by minimizing the steps required for usual treatment, especially in case of dairy wastewater treatment, where the pH is first neutralized with some chemicals and then additional treatments/steps are done.
Effect of incubation temperature on lipase production
Temperature is one of the most important physical factors for growth and enzyme production. To check the optimum temperature in the present case, incubation was carried out at different temperatures in the range of 15 to 40Â°C. It is clear from Fig. 3(b) that lipase production increased with increasing temperature with maximum production at 30Â°C. Further increase in temperature resulted in decreased enzyme activity showing 38% and 28.2% activities at 35 and 40°C respectively. While at 25°C, 88% lipase activity was detected. Singh et al.20 and Kanwar et al.23 have also reported optimized temperature of 30Â°C and 34Â°C for lipase production by newly isolated strain of Pseudomonas aeruginosa and Pseudomonas sp. respectively.
Effect of additional carbon and nitrogen sources
Carbon and nitrogen source is one of the important factors for lipase production as lipases are by and large inducible enzymes.2 Among nitrogen sources, generally organic nitrogen source like peptone and yeast extract, are preferred for lipase production while triacylglycerols, fatty acids, sugars, sugar alcohol and polysaccharides as carbon sources.2 Effect of added carbon and nitrogen sources on lipase production was tested in case of Aeromonas sp. S1 also. Since the fermentation in this study was carried out in nutrient rich media with enough carbon and nitrogen source, and additional sources also available from SDOCE. This might be the reason that supplementation of extra carbon and nitrogen sources did not enhance the lipase yield by Aeromonas sp. S1. Among the carbon sources explored, Mustard oil (1% v/v) did not affect the lipase production; however 43% of lipase activity was observed in presence of coconut oil (1% v/v) as compared to control (Fig. 4(a)). Other carbon sources like maltose (1% w/w) and glucose (1% w/w) showed inhibitory effect, resulting in 17 and 5.5% of lipase production respectively compared to control. The inhibitory nature of glucose and maltose on lipase production by Bacillus sp. strain 42 was also reported by Eltaweel et al.24. Li et al.25 showed the lower production of lipase from Acinetobacter radioresistens using olive oil and suggested that its olic acid suppressed lipase synthesis. Inhibitory and inducible properties of triglycerides on lipase production were also suggested by Immanuel et al. 18
Because most of the organic nitrogen sources were available from the media, the effect of different inorganic nitrogen sources (1% w/w) viz. sodium nitrate, ammonium chloride and ammonium sulphate was tested on lipase production by Aeromonas sp. S1. Lipase production was found to be inhibited with supplemented inorganic nitrogen sources as shown in Fig. 4(b). There was 83, 78 and 40% of lipase activity in the presence of sodium nitrate, ammonium sulphate and ammonium chloride respectively. The inhibitory effect of sodium nitrate and other inorganic nitrogen sources are also reported by Eltaweel et al.24 in case of lipase production by Bacillus sp. Strain 42. However, no detectable lipase activity could be determined in case of Bacillus megaterium AKG-in the presence of ammonium chloride as nitrogen source.26
The optimized culture conditions for lipase production from Aeromonas sp. S1 were established as following: Nutrient media containing 3% (v/v) SDOCE, pH 8.0 and incubation temperature of 30Â°C.
Under all optimized conditions, the lipase yield was enhanced to 24-fold (195 Uml-1) at 24 h of growth compared to initial 8.13 Uml-1 at 24 h. This level of lipase production from Aeromonas sp. S1 by using SDOCE as additive nutrient source was comparable to that obtained with other natural substrate under submerged fermentation. For instance, Lotrakul and Dharmsthiti16 have reported 450 Uml-1 of lipase production by Aeromonas sobria in the presence of soybean meal and whey. Similarly combination of brewery co product, yeast extract, malt extract, Tween 80 and olive oil with cheese whey provided an average increase of 15 U ml-1 in the lipase activity using Candida rugosa, through experimental design and response surface methodology.27
Since this lipase preparation was required for dairy wastewater treatment, it was worthwhile to look for its enzymatic characteristics.
The optimum pH determined for lipase activity was in the range of 9.0 to 10.0, with maximum activity at pH 10.0. At higher pH value of 11.0, the enzyme activity was 70 % of the maximal activity at pH 10.0 (data not shown). Lipase was found to be most active at 55Â°C, though it showed considerable activity over the range of 35 - 45°C. While the lipase activity was reduced to 32.3% and 22.7% at 65Â°C and 75Â°C respectively (data not shown). Similar pH and temperature optima were also reported in case of B. licheniformis strain H1.28 Bacillus alcalophilus shows optimum pH of 10.6 and temperature of 60Â°C.29 The optimum temperature of lipase produced by Aeromonas sorbia LP004 was found to be 45°C and was highly stable in alkaline conditions up to 9.5 pH.16
Preliminary treatment of dairy wastewater
Dairy wastewater contains high concentration of lipids in the form of O&G and proteins that present low biodegradability and can cause severe environmental pollution if not treated properly. High concentration of O&G often causes problems in the various wastewater treatment processes also. According to Cammarota and Freire 4 and Alberton et al.5, in the activated sludge process, high O&G levels lead to the formation of films around the biological flocs, hindering the transfer of oxygen and substrate to the floc microorganisms and leading to the proliferation of filamentous microorganisms. In the case of anaerobic digestion, excessive amounts of O&G inhibit the action of acetogenic and methanogenic bacteria.5 Such problems necessitate the pretreatment of wastewater for removal of O&G. Various physicochemical methods are being employed for such pretreatment. However, in the recent years use of microbial cultures and enzymes for increasing hydrolysis during or prior to biological treatment process 4, 30, 31 is the topic of attention due to its sustainable and environment-friendly approach. Such pretreatment methods generally consist of the cultivation of the lipase producing microbial strains in the effluents or direct addition of crude or pure lipase preparations (fermented solid or liquid media containing enzyme) to the sample. In the present study, treatment of dairy wastewater was carried out by using both the methods individually; by inoculation of lipase producing Aeromonas sp. S1 as well as by addition of its crude lipase preparation. A control set was also set up in which no culture or enzyme preparation was added extraneously.
The wastewater used in the present study was collected from a local dairy industry, New Delhi, India. The physico-chemical and biological analysis of the sample presented the following characteristics: pH = 10; temperature = 20°C; COD = 1200Â±200 mgl-1; BOD = 850Â±50 mgl-1; TSS = 425Â±62 mgl-1; O&G = 219Â±27 mgl-1; Total nitrogen = 20.2 mgl-1; Total phosphorus = 2.4 mgl-1 and Total coliforms= 7500 MPN 100ml-1. As the sample was found to contain higher value of COD, TSS, and O&G as compared to Indian standard limits for the dairy effluent discharge, 32, 33 its treatment before disposal is required. Similar characteristics of dairy wastewater with COD, O&G (Total fat content) and TSS values of 1792 mgl-1, 360 mgl-1 and 320 mgl-1 respectively is reported by Davery et al.33
Microbial treatment of dairy wastewater
The potency of Aeromonas sp. S1 was evaluated for reduction of COD, O&G and TSS from dairy wastewater. Fig. 5(a) clearly shows that there is remarkable decrease in the COD, O&G and TSS contents after treatment with Aeromonas sp. S1. The strain could reduce the COD and TSS of wastewater by 93% and 47% respectively after 96 h of treatment. The O&G content was also found to be reduced up to 75% of initial value after 96 h. The reduction in value of BOD and O&G from dairy wastewater to dischargeable limits using different individual microbes and in combination as consortia was also reported by Prasad and Manjunath.34 The formulated mixture of microbes (P. aeruginosa LP602, Bacillus sp. B304, Acinetobacter calcoaceticus LP009) reported by Prasad and Manjunath,34 could effectively reduced initial 3200 mgl-1 BOD and 25000 mgl-1 lipid content to 40 and 80 mgl-1 respectively during 12 day aerobic incubation at 30Â°C and 200 rpm. The fast attainment of dischargeable values for COD in case of Aeromonas sp. S1 clearly shows the efficacy of the isolate (Table 1.). Mongkolthanaruk and Dharmsthiti35 also reported the use of mixed culture composed of P. aeruginosa LP602, Acinetobacter calcoaceticus LP009 (both lipase-producing bacteria) and Bacillus sp. B304 (an amylase and protease producing bacterium) for the treatment of lipid rich wastewater.
Enzymatic treatment of dairy waste water
High lipase activity is a critical prerequisite, if the enzymes are to be applied for industrial effluent treatment 4. Lipase preparation containing 195 U ml-1 of initial lipase activity was used for the treatment of wastewater. Fig. 5(a) shows that there was significant reduction in COD, TSS and O&G compared to control after treatment with this enzymatic preparation. Since in case of enzymatic treatment, there was 86% reduction of COD in 24 h (Fig. 5(a)), and thereafter it remained almost constant till 96 h, an experiment was set-up to check earlier COD removal as enzymes are known to be fast in their reactions. Samples were withdrawn every 4 h interval during the treatment. The reduction of 44 and 69% of COD was observed in first 4 and 8 h respectively (Fig. 5(b)). In 12 h of treatment, COD reduction reached to 86 % which remained constant in subsequent time interval of 24, 48 and 96 h. This experiment confirmed that enzymatic treatment was very fast as compared to the microbial treatment where only 71.5% of COD reduction was observed after 24 h of treatment. There was 75% and 45% reduction of O&G and TSS respectively after 96 h of Lipase treatment.
Similar enzymatic pretreatment using fermented solid containing the lipases of Rhizopus microsporus was used for pretreatment of dairy wastewater by Alberton et al.5 They evaluated the efficacy of their pretreatment on the basis of BOD, COD and O&G analyses. During 72 h of treatment, the initial COD of 6908 mgl-1 was reduced to 2570 mgl-1 using solid fermented media containing lipase. The O&G content was also reduced to 250 mgl-1 during 72 h of treatment from initial value of 1300 mgl-1. They have supplemented extra dairy fat to wastewater; initially it contained 400-600 mgl-1 of O&G.
As shown in Fig. 5(a), there was reduction in the control set (without addition of extraneous culture or enzyme) also. Since control flask was also kept under similar conditions like test sample i.e. with shaking of 200 rpm. This shaking might had provided enough oxygen and mixing conditions for growth of indigenous microbes of the wastewater, resulting in COD, TSS and O&G reduction. To confirm this, another control flask was also kept under similar experimental conditions but without shaking. The samples were withdrawn at 0 and 96 h, and surprisingly, there was no reduction of O&G, TSS and negligible reduction of COD (less than 5%). This supported the previous assumption that due to shaking, it was the growth of indigenous microorganisms that resulted in reduction of organic matter (COD).
The residual O&G and TSS values obtained in this study after 96 h of microbial and enzymatic treatments were closer to dischargeable value32 and for COD; the value is under dischargeable limit33 as shown in Table.1. The above results show that both Aeromonas sp. S1 and its lipase could effectively be used for bioremediation of dairy wastewater.
The results obtained show that natural substrate in the form of SDOCE have a good inducer property for lipase synthesis. Thus it might be worthwhile to use this natural by-product as a cost-effective source for lipase production. The isolate Aeromonas sp. S1 and its lipase also found to be promising for bioremediation of lipid rich wastewater originating from food processing industries.