ABSTRACT

Paper and pulp mill is a source of major pollution generating industry leaving huge amount of intensely colored effluent to the receiving end. Rapid increase of population and the increased demand for industrial establishments to meet human needs have created problems such as over exploitation of available resources, increased pollution taking place on land, air and water environment. The intention of this research paper is to identify predominant bacteria and fungi in paper and pulp mill effluent in addition to evaluate the degradation efficiency of individual isolates and combination of isolates. Treatment efficiency of individual isolates and combination of isolates are evaluated by shake flask method. Combination of Pseudomonas Alkaligenes, Bacillus subtilis along with Tricho derma reesei shows higher BOD, COD reduction of 99% and 85% respectively. As individual isolates Pseudomonas Alkaligenes show 92% BOD reduction and 77% COD reduction over other bacterial isolates and Trichoderma reesei removed 99% BOD and 80% COD respectively.

1. Introduction

Paper and pulp mill is a large industrial enterprise that generates a significant amount of wastewater containing high concentration of lignin causing brown color and high Chemical Oxygen Demand. There has been considerable organic matter in pulp mill effluents on the environment. Some members of this family are known to be toxic, mutagenic, persist ant and bioaccumulating and are thought to cause numerous harmful disturbances in biological systems. There is no industry which does not add wastes into the environment. The introduction of contaminants through effluent and sludge to different environmental compartments can often overhelm the self cleansing capacity of recipient ecosystems and thus result in the accumulation of pollutants to problematic or even harmful levels. Prabu and Udayasoorian [1] reported that white rot fungus isolated from soil samples enriched by continuous pulp and paper mill effluent irrigation and identified as Phanerochaete chrysosporium was capable of 84% effluent decolourization along with 79% COD reduction.

Over several decades attempts have been made to remove the dark color from the effluents. Of late industry follows either chemical oxidation/precipitation methods or biological methods for color removal. Chemical oxidation/precipitation methods are tedious, provide an additional environmental load. Biological methods are often preferred since it has many advantages like rapid biodegradation rates, low sludge yield and excellent process stability. Biological methods are of particular interest because they can also reduce chemical and biological demands (COD, BOD), which are also significant problem in pulp wastewater and so reduce holding times in aeration and sedimentation ponds prior to wastewater discharge into the environment [2-4].

A number of research studies have discovered that a group of extracellular isoenzymes called ligninases which are lignin peroxidase (LiP), manganese-dependent peroxidase (MnP) and laccase produced by some microorganisms are capable of degrading lignin present in the paper and pulp mill effluent. de Oliveria et al. [5] evaluated the ability of these bacterias to remove color and COD from paper mill effluent. Bacillus pumilus CBMAI 0008 isolated from wood decomposition and Paenibacillus sp CBMAI 868 isolated from paper mill wastewater are able to produce alkaline enzymes under thermophilic conditions, including xylanases and manganese dependent peroxidase.

Some investigators have made an attempt to treat pulp and paper mill wastewater by the thermophilic temperature at which they are discharged to reduce energy cost for treatment. Reddy et al. [6] treated the pulp and paper mill effluent by using thermophilic microorganisms in batch systems. They compared the aerobic treatment of pulp and paper mill effluent at the temperatures of 40°C, 50°C and 60°C. Maximum removal efficiency of 55.2% was achieved at 40ºC. However once degradation is obtained the degradation rate decreases significantly as temperature increases. Ruiz-Ordaz et al. [7] investigated on phenol biodegradation using repeated batch culture of Candida tropicalis in a multistage bubble column. The phenol removal efficiency of 98.7% was achieved. Marihal et al. [8] utilized Rhizobacteria isolated from pentachlorophenol-tolerant crop species for biodegradation of pentachlorophenol. 90% phenol reduction was achieved using these bacteria. Pentachlorophenol (PCP) is a polychlorinated aromatic compound that is widespread in industrial effluents and is considered to be a serious pollutant. PCP is also formed unintentionally in effluents of paper and pulp industries. A number of plant species were evaluated for their ability to tolerate different concentrations of Pentachlorophenol (PCP) in the soil. An important strategy for effluent treatment is the isolation and characterization of genetically significant microorganisms together with designing and optimization of process parameters to deal with specific environment pollutants. Nagarathnama et al. [9] successfully treated kraft mill effluent using Rhizopus oryzae. Rhizopus oryzae, a zygomycete, decolorize, dechlorinate and detoxify bleach plant effluent at lower cosubstrate concentration. With glucose at g/L, this fungus removed 92- 95% of the color, 50% of the chemical oxygen demand, 72% of the adsorbable organic halide and 37% of the extractable organic halide in 24 hour at temperatures of 25-45ºC and a pH of 3-5. The aim of this research study is to begin with isolating the predominant fungi and bacteria present in pulp and paper mill effluent and evaluating the degradation efficiency of individual isolates and combination of isolates in laboratory scale.

2. Materials and Methods

2.1. Effluent Source

The effluent was obtained from SESHASAYEE PAPER MILL, Erode, Tamilnadu, a South Indian based Integrated Pulp and Paper Industry. The paper pulp effluent from the inlet, outlet of primary settling tank was used for investigation. The sample was collected using a sterile plastic container and transported to the RND Softech Private Limited research laboratory situated in Coimbatore within 4 hours. The effluent was stored at 4ºC until required.

2.2. Methods

All the testing was performed according to Standard Microbiological methods for the Examination of Water and Wastewater as described [10]. Shake flask method was used to evaluate the treatment efficiency of individual isolates and combination of isolates.

2.3. Isolation and Identification of Predominant Microorganisms

The sample was serially diluted using sterile pipettes from 10^-1 to 10^-8 dilution. Figure 1 shows bacterial and fungal strain capable of growing on Nutrient agar and PDA agar medium. Five different types of bacteria were predominant in the raw effluent; single type of fungi was found to be present. For enumeration of bacteria nutrient agar medium containing peptone (5 g/L), yeast extract (1.5 g/L), sodium chloride (5 g/L), agar (15 g/L) and for enumeration of fungi Potato dextrose agar containing potato (200 gm), dextrose (20 gm, agar-15 gm, distilled water (1000 mL) at pH 5.6 was used. To obtain pure culture the cultures were repeatedly streaked nutrient agar medium and incubated at 37°C for 24 hrs. The isolated bacteria were identified by colony morphology, gram staining, microscopic observation and confirmation test. The identified bacteria were Pseudomonas alkaligenes, Bacillus pumilus, Bacillus subtilis, Klebsielle sp, Proteus sp. The isolated fungal culture was identified as Trichoderma reesei using Lactophenol cotton blue staining method.

3. Microbial Treatment of Effluent

The raw effluent physiochemical parameters are given in Table 1. The raw effluent was treated using the isolated bacteria and fungi. The treatment efficiency was validated by calculating the percentage reduction of physiochemical parameters.

Table 2 gives the treatment efficiency of the five isolates for comparison and the isolates Klebsielle sp, Proteus sp, are excluded from further study as their percentage reduction of parameters are low compared to other three isolates.