Biological Approach for the Treatment of Pulp and Paper Industry Effluent in Sequence Batch Reactor

Pulp and paper industrial effluent is rich in recalcitrant compounds and causes pollution. For the treatment of such compounds activated sludge process is frequently used in which F/M ratio is kept low. This treatment results in effective biochemical oxygen demand removal but other waste water parameters are not reduced effectively due to lack of dissolve oxygen. In the present study sequential batch reactor was used for the removal of pollutants from the waste water of pulp and paper mill by using bacterial consortium (Klebsiella sp., Alcaligens sp. and Cronobacter sp.). The aim of present research is to identify the influences of F/M ratio and dissolved oxygen concentration on the microorganism’s growth and pollutant removal. The process of bioremediation was optimized by Taguchi approach. Bioremediation experiment resulted in reduction of chemical and biochemical oxygen demand up to 72.3% and 91.1%, respectively. A significant reduction in colour (55%), adsorbable organic halides (45.4%), total dissolve solids (22%) and total suspended solids (86.7%) was also observed within 14hrs while, the sludge volume index was 52. The wastewater after the treatment process meets the standard given by regulatory agencies and can be discharged into the environment without any risks. Citation: Kumar V, Dhall P, Naithani S, Kumar A, Kumar R (2014) Biological Approach for the Treatment of Pulp and Paper Industry Effluent in Sequence Batch Reactor. J Bioremed Biodeg 5: 218. doi:10.4172/2155-6199.1000218 Volume 5 • Issue 3 • 1000218 J Bioremed Biodeg ISSN: 2155-6199 JBRBD, an open access journal Page 2 of 10 plant with high mixed liquor suspended solids (MLSS) in order to buffer the BOD, pH, waste water composition or temperature. The manufacturing process depends upon the demand therefore the raw material changes accordingly, which in turn changes the ultimate pollution load in the wastewater. If the MLSS of the tank is high it can tolerate the sudden shock. This treatment results in effective Biochemical Oxygen Demand (BOD) removal but face several problems. Low F/M ratio leads inadequate food for the population of microorganisms and problem arises in maintaining the sufficient dissolved oxygen concentration. This ultimately leads to enhanced growth of filamentous microorganisms. However, such systems are generally less effective in removing colour, COD and chlorinated phenolic compounds [19]. Therefore, more advanced alternative biological wastewater treatment strategies will be required for the treatment of effluent holistically and meet the new and more stringent discharge limits set for paper industry effluent. Such new biological treatment technologies must be developed to degrade lingo-cellulosic compounds and halogenated chemicals in effluents that pose the greatest threat to human health. Mill description: The mill selected for the study is 50 year old and has capacity of 200 ton/day of paper production, situated in western Uttar Pradesh, India (29.9640°N, 77.5460°E). Kraft pulping process is the initial step followed oxygen delignification after that the chemical bleaching, is carried out in 3 steps (i) chlorination (C) (ii) extraction (E) (iii) hypochlorite treatment (H). Each step is preceded and followed by countercurrent washing with fresh water. A total of 25,000 KLD of water is consumed for the entire process of pulping and paper making whereas, around 23,000 m3/day is discharged as wastewater. In the present study sequential batch reactor was used for the removal of pollutants from the waste water of pulp and paper mill by using bacterial consortium having potential to degrade lingo-cellulosic compounds. The aim of present research is to treat the pulp and paper effluent holistically in environmental friendly manner and also to know the influences of F/M ratio and Dissolve Oxygen (DO) concentration on the microorganism’s growth and pollutant removal. Materials and Methods Chemicals and consumables All the chemicals were of analytical grade and procured from Merck. Kraft lignin (KL), Carboxy Methyl Cellulose (CMC), birch wood xylan was procured from Sigma Aldrich. All the solutions were prepared in milli-Q water and were preserved in SCHOTT duran bottle. Media were procured from Himedia and glassware's and plastic wares were purchased from Rivera Pvt. Ltd. Waste water sample collection and characteristics Waste water sample collection: The samples were collected in bottles with at least 2.5 cm empty at neck to facilitate mixing by shaking, before examination [20]. For the present study, effluent samples were collected from over flow of primary clarifier (containing combined effluent of whole process) of large scale paper mill situated in UP, India. For collecting the samples composite sampling was done. The samples were collected in clean, plastic jerry can (container). Samples were transported within 4hr under refrigerated conditions and stored in cold room at 4°C till further use. Collection of soil sample: Samples were collected on 5th May 2010 from the industrial premises. Soil samples (3 in number) were collected from the upper layer (2 cm depth) of the soil where most of the microbial activity takes place. Three areas, Wood Yard Section (WYS), Combined Bleach Effluent discharged point (CBE) and Final Discharge Point (FDP) were selected for the sample collection. Form each area three random samples were collected. Those random samples were mixed to get one sample from each area. All the samples were collected in plastic bags. After collection the bags were kept at 4°C till further use. Characteristics of waste water: The effluent was characterized for various physico-chemical parameters like: pH, color, lignin, COD, BOD, Total Dissolved Solids (TDS), Total Suspended Solids (TSS), AOX and each parameter was analyzed in triplicate in order to see the standard error in handling. All analyses were carried out as per standard procedures [20]. Color was measured by the spectroscopic method (National Council for Air and Stream Improvement-NCASI Method 71.01.1999) [21]. AOX of the effluents was measured using the procedure standardized in Germany in 1985 and known as DIN 38 409 Tail 14 AOX procedure. Isolation of autochthonous bacteria Bacteria were isolated from the pulp and paper mill premises. Three samples were collected (i) from wood yard section, (ii) combined bleach effluent discharged point, (iii) soil from near the final discharge point. The pulp and paper waste water is rich in lignin and other chemicals. The areas for collecting the soil samples were selected by assuming the presence of delignifying bacteria in that sector with recalcitrant compounds and genetic potency to degrade these compounds. Isolation was done by using such soil samples because it is easy to acclimatize those bacteria in the paper industry effluent due to their existence in particular environment and these bacteria can be enriched in the presence of toxic compounds therefore, a significant strains will be evolved with the process of acclimatization. Serial dilution of enrichment culture (supplementary Table 1) was done after 45 days of incubation at 150 rpm and 35°C. For each soil sample three enrichment flasks were prepared containing different substrate i.e., lignin, Carboxymethyl Cellulose (CMC) and birch wood xylan. Different media were prepared in order to isolate the bacteria which will able to degrade lingo-cellulosic compounds (supplementary Table 2). For preparing the bacteria for the task, sub-culturing was done in the enrichment media after every 15 days for 3 times. After serial dilution the 50 μl of the dilution was spread on the agar plates of minimal salt medium (MSM) containing lignin/cellulose/ xylan as sole carbon and energy source. These plates were incubated at 35°C for 7 days and bacteria were purified at nutrient agar plates. Plates were preserved at low temperature till further use. Screening of selected isolates The screening method included functional assays for the presence of lingo-cellulosic compound degrading enzymes. Decolourization of lignin-mimicking dyes was assessed in agar plates. The plates were prepared by autoclaving the nutrient agar at 15 psi for 20 min. The following dyes were selected: AB: Azure-B; BB: Bromophenol Blue; MB: Methylene Blue; PR: Phenol Red; MG: Malachite Green. Dye was added individually 50 mg/l of media and poured in to the plates. After pouring, the plates were kept for solidification at room temperature. Sterilized disc borer was used to cut the disc of approximately 1 cm (dia) size in solidify plates. Bacterial culture supernatant (200 μl) was poured into the well. The plates were incubated at 35°C and monitored daily for 3 days. Bacterial isolates which produced a visible change in the colour of substrate were selected for bioremediation study. Citation: Kumar V, Dhall P, Naithani S, Kumar A, Kumar R (2014) Biological Approach for the Treatment of Pulp and Paper Industry Effluent in Sequence Batch Reactor. J Bioremed Biodeg 5: 218. doi:10.4172/2155-6199.1000218 Volume 5 • Issue 3 • 1000218 J Bioremed Biodeg ISSN: 2155-6199 JBRBD, an open access journal Page 3 of 10 16–18 h. The culture was harvested by centrifugation at 4°C and 7000 rpm followed by washing twice with sodium phosphate buffer (pH 6.8–7.0). The supernatant was discarded and pellets were stored for the further experiments. At the time of experiment, different pellets were re-suspended according to the 4 consortia designed and inoculated in the wastewater of pulp and paper industry, the effluent sample was supplemented with nutrients N and P in the form of urea and diammonium hydrogen phosphate in the ratio of (BOD) 100: nitrogen (N) 5: phosphorus (P) 1 and the flasks were kept in shaking incubator at 150 rpm and 35°C for 24 h. After incubation, Chemical Oxygen Demand (COD) was estimated according to the procedure mentioned in standard methods, American Public Health Association (APHA). The obtained results were compared with the control flask which is Formulation and screening of bacterial consortia Pulp and paper wastewater is complex and toxic in nature so, it is difficult for single microorganism to degrade it effectively. Therefore, selected single isolates were used for the formulation of different consortia. Each consortium consists of three bacteria. Total four consortia were formulated by random combination isolates (supplementary Table 3). Different cultures were inoculated in 25 mL of nutrient broth (NB) and incubated overnight at 35°C and 150 rpm. The mother cultures were checked by streaking on nutrient agar plates which were then incubated at 35°C for 16-18 hrs. These mother cultures were used for sub culturing. 100 μl of culture was inoculated into 100 mL of NB and incubated at 35°C under shaking conditions for a period of Exp. No. P1 P2 P3 P4 P5 P6 Percentage reduction in COD S/N Ratio Temp pH F/M DO Time NP T1 T2 T3 1 25 6 0.5 0.5 14 100:6:1.5 55 60 58 27.2 2 25 6 1 1 16 100:5:1 67 65 67 28.41 3 25 6 1.5 1.5 18 100:4:0.5 57 61 59 29.83 4 25 7 0.5 0.5 16 100:5:1 68 65 72 25.77 5 25 7 1 1 18 100:4:0.5 65 69 71 26.98 6 25 7 1.5 1.5 14 100:6:1.5 59 65 61 26.09 7 30 8 0.5 1 14 100:4:0.5 71 65 69 26.98 8 30 8 1 1.5 16 100:6:1.5 64 67 67 28.6 9 30 8 1.5 0.5 18 100:5:1 71 67 63 24.47 10 30 6 0.5 1.5 18 100:5:1 64 69 72 24.54 11 30 6 1 0.5 14 100:4:0.5 69 71 74 29.04 12 30 6 1.5 1 16 100:6:1.5 64 69 64 27.13 13 35 7 0.5 1 18 100:6:1.5 72 67 70 28.84 14 35 7 1 1.5 14 100:5:1 74 73 76 33.74 15 35 7 1.5 0.5 16 100:4:0.5 58 69 64 21.23 16 35 8 0.5 1.5 16 100:4:0.5 69 71 64 25.5 17 35 8 1 0.5 18 100:6:1.5 72 69 74 29.09 18 35 8 1.5 1 14 100:5:1 65 68 71 27.1 Table 1: Results of trials carried out for different experiments designed using Taguchi approach and their S/N ratios. T(°C) pH COD (mgL-1) BOD (mgL-1) TDS (mgL-1) TSS (mgL-1) AOX (mgL-1) Color (PCU) Winter 20-29 7-8.5 600-1000 250 35