Bioremediation of paper and pulp mill effluents

Pulp and paper mill effluents pollute water, air and soil, causing a major threat to the environment. Several methods have been attempted by various researchers throughout the world for the removal of colour from pulp and paper mill effluents. The biological colour removal process uses several classes of microorganisms--bacteria, algae and fungi--to degrade the polymeric lignin derived chromophoric material. White rot fungi such as Phanerochaete chrysosporium, Corius versicolor, Trametes versicolor etc., are efficient in decolourizing paper and pulp mill effluents. Gliocladium virens, a saprophytic soil fungus decolourised paper and pulp mill effluents by 42% due to the production of hemicellulase, lignin peroxidase, manganese peroxidase and laccase.     

Decolorization of pulp and paper mill effluent by growth ofAspergillus niger

Pulp and paper mill effluent was decolorized by growth ofAspergillus niger. Adding glucose (2.0 g/l) and NH₄H₂PO₄ (1.0 g/l) improved decolorization by the fungus (leaving 19% of original colour) and reduced the BOD₅ (43%) and the COD (41%) of the effluent after 48 h of incubation.

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Résumé L'effluent d'un atelier de pâte à papier a été décoloré par la croissance d'Aspergillus niger. L'ajout de glucose (2.0 g/l) et de NH₄H₂PO₄ (1.0 g/l) a amélioré la décoloration de l'effluent par la molsissure, lalssant 19% de la couleur originale, réduisant la DBO₅ de 43% et la DCO de 41%, après 48 h d'incubation.

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This work was carried out at the Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore-641 003, India.     

The treatment of pulp and paper mill effluent: a review

The manufacture of paper generates significant quantities of wastewater; as high as 60 m3/tonne of paper produced. The raw wastewaters from paper and board mills can be potentially very polluting. Indeed, a recent survey within the UK industry has found that their chemical oxygen demands can be as high as 11000 mg/l. This paper reviews the processes involved in paper making and examines the effects which they could have on the environment. It also evaluates the treatment processes which are used to minimise these effects. In line with the majority of UK practice, it focuses mainly on aerobic biological treatment and, in particular, on the activated sludge process. This means that there is an in-depth discussion about the problems associated with filamentous bacteria and sludge "bulking". The paper also discusses the way in which anaerobic digestion can be applied to the treatment of liquid wastes from the manufacture of paper.     

造纸废水纸浆沉淀物中未培养微生物纤维素酶基因的克隆和鉴定

提取纯化造纸废水纸浆沉淀物的宏基因组DNA并构建16S rDNA文库,系统发育分析显示该环境中存在大量的未培养细菌且具有种类的多样性。以柯斯质粒为载体构建了1个含10000个克隆的宏基因组文库,文库容量为3.53×108bp。筛选文库得到2个表达内切葡聚糖酶活性的克隆、3个表达外切葡聚糖酶活性的克隆和2个表达β-葡萄糖苷酶活性的克隆。从表达不同活性的克隆中分别挑选活性最强的进行鉴定,得到3个新的纤维素酶基因umcel5L、umcel5M和umbgl3D。umcel5L、umcel5M和umbgl3D分别编码产生内切葡聚糖酶、纤维糊精酶和β-葡萄糖苷酶,其编码产物与已报道的纤维素酶一致性最高的分别为43%、48%和46%。这是第一次采用未培养方法对造纸废水纸浆沉淀物中的细菌多样性进行分析并从中克隆纤维素酶基因的报道。     

Cloning and identification of cellulase genes from uncultured microorganisms in pulp sediments from paper mill effluent]

The metagenomic DNA of pulp sediments from paper mill effluent was extracted and purified. The 16S rDNA was amplified using the purified metagenomic DNA as template and a 16S rDNA library was prepared. Sequence analysis of 16S rDNA clones showed that diverse of uncultured bacteria inhabit in this environment, which can be classified into 4 clusters as Spirochaetes, Proteobacteria, Bacteroidetes and Firmicutes. A metagenomic library containing 10000 clones was constructed into cosmid vector, and the capacity of inserted DNA of which was 3.53 x 10(8) bp. Functional screening of the library resulted in isolation of two independent clones expressing endoglucanase activity, three independent clones expressing exoglucanase activity and two independent clones expressing beta-glucosidase activity. One clone expressing strongest enzyme activity from each activity category was chosen to be further analyzed. Three novel cellulase genes designated as umcel5L, umcel5M and umbgl3D were identified by subcloning, sequencing and expression. The umcel5L encodes an endoglucanase belonging to glycosyl hydrolase family 5, which is most related to an endoglucanase from Bradyrhizobium japonicum at 43% identity and 59% similarity. The umcel5M encodes a cellodextrinase belonging to glycosyl hydrolase family 5, which is most similar to a cellodextrinase from Fibrobacter succinogenes at 48% identity and 69% similarity. The umbgl3D encodes a putative beta-glucosidase belonging to glycosyl hydrolase family 3, which shares highest homology with a beta-glucosidase from Thermotoga maritima at 46% identity and 61% similarity. It is the first time to reveal the bacterial diversity of pulp sediments from paper mill effluent and clone novel cellulase genes from the bacteria by culture-independent method.     

Coliform bacteria and nitrogen fixation in pulp and paper mill effluent treatment systems

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N(2) fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N(2) fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N(2)-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N(2) fixation (0.87 to 4.90 mg of N liter(-1) day(-1)) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.     

Colour removal of anaerobically treated pulp and paper mill effluent by microorganisms in two steps bioreactor

In the present study sequential anaerobic and aerobic treatment in two steps bioreactor was performed for removal of colour in the pulp and paper mill effluent. In anaerobic treatment, colour (70%), lignin (25%), COD (42%), AOX (15%) and phenol (39%) were reduced in 15 days. The anaerobically treated effluent was separately applied in bioreactor in presence of fungal strain, Paecilomyces sp., and bacterial strain, Microbrevis luteum. Data of study indicated reduction in colour (95%), AOX (67%), lignin (86%), COD (88%) and phenol (63%) by Paecilomyces sp. where as M. luteum showed removal in colour (76%), lignin (69%), COD (75%) AOX (82%) and phenol (93%) by day third when 7 days anaerobically treated effluent was further treated by aerobic microorganisms. Change in pH of the effluent, and increase in biomass of microorganisms substantiated results of the study, which was concomitant to the treatment method.     

Reduction of (per)chlorate by a novel organism isolated from paper mill waste

As part of a study on the microbiology of chlorate reduction, several new dissimilatory chlorate-reducing bacteria were isolated from a broad diversity of environments. One of these, strain CKB, was selected for a more complete characterization. Strain CKB was enriched and isolated from paper mill waste with acetate as the sole electron donor and chlorate as the sole electron acceptor. Strain CKB is a completely oxidizing, non-fermentative, Gram-negative, facultative anaerobe. Cells of strain CKB are 0.5 x 2 microm and are highly motile, with a single polar flagellum. In addition to acetate, strain CKB can use propionate, butyrate, lactate, succinate, fumarate, malate or yeast extract as electron donors, with chlorate as the sole electron acceptor. Strain CKB can also couple chlorate reduction to the oxidation of ferrous iron, sulphide, or the reduced form of the humic substances analogue 2,6-anthrahydroquinone disulphonate. Fe(II) is oxidized to insoluble amorphous Fe(II) oxide, whereas sulphide is oxidized to elemental sulphur. Growth is not associated with this metabolism, even when small quantities of acetate are added as a potential carbon source. In addition to chlorate, strain CKB can also couple acetate oxidation to the reduction of oxygen or perchlorate. Chlorate is completely reduced to chloride. Strain CKB has an optimum temperature of 35 degrees C, a pH optimum of 7.5 and a salinity optimum of 1% NaCl. Strain CKB can grow in chlorate and perchlorate concentrations of 80 or 20 mM respectively. Under anaerobic conditions, strain CKB can dismutate chlorite into chloride and O2, and is only the second organism shown to be capable of this metabolism. Oxidized minus reduced spectra of whole-cell suspensions of strain CKB showed absorbance maxima at 423, 523 and 552nm, which are indicative of the presence of c-type cytochrome(s). Analysis of the complete sequence of the 16S rDNA indicates that strain CKB is a member of the beta subclass of the Proteobacteria. The phototroph Rhodocyclus tenuis is the closest known relative. When tested, strain CKB could not grow by phototrophy and did not contain bacteriochlorophyll. Phenotypically and phylogenetically, strain CKB differs from all other described bacteria and represents the type strain of a new genus and species.     

Aerobic biodegradation of 1,2-dichloroethane and 1,3-dichloropropene by bacteria isolated from a pulp mill wastewater effluent in South Africa

Large volumes of chlorinated aliphatic hydrocarbons are produced annually for a variety of industrial and commercial uses. They therefore constitute common contaminants of soil and groundwater causing serious environmental and human health problems. In this study, three bacteria were isolated from a pulp mill wastewater effluent in South Africa by culture enrichment technique and characterized for their ability to degrade 1,2-dichloroethane (1,2-DCE) and 1,3-dichloropropene (1,3-DCP). Specific growth rate constants of the organisms ranged between 0.864∼1.094 and 0.530∼0.585 d⁻¹ in 1.2-DCE and 1,3-DCP, respectively, while the degradation rate constant of the compounds ranged variously between 0.33 and 1.006 d⁻¹, with 1,2-DCE generally better utilized than 1,3-DCP. Gas chromatographic analysis revealed up to 75 and 80% removal of 1,2-DCE and 1,3-DCP, respectively, above that observed in the control bottles. These organisms also demonstrated high haloalkane dehalogenase activities with specific dehalogenase activities ranging between 0.25∼0.31 U (mg protein)⁻¹. Analysis of their 16S rRNA gene sequences revealed that they belong to the generaPaenibacillus, Bacillus, andMicrobacterium.     

Genetic and metabolic diversity of streptomycetes in pulp and paper mill effluent treated crop fields

Irrigation of farm field with water mixed with pulp and paper mill effluent from Century pulp and paper mill in Uttrakhand state of India for over last 25 years in succession increased streptomycetes population (120 × 10⁵) compared to the fresh water irrigated fields (48 × 10³ in WIF). Denaturing gradient gel electrophoresis, amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, BIOLOG™ substrate usage, production of extracellular enzymes (xylanase and cellulase) and plant growth promoting attributes were applied to monitor changes in genetic and metabolic diversity of streptomycetes. Significant variation was observed for production of extracellular enzymes, Indolic compounds, siderophore and P-solubilisation among isolates. Metabolic substrate usage of Streptomyces isolates was evaluated using the BIOLOG™ GP2 plates and unique carbon substrate usage profiles were observed. Based on 16S rRNA gene sequencing, the isolates were identified as Streptomyces variabilis, Streptomyces spp. S. glaucescens, S. viridochromogenes, S. cinnabarinus, S. aburaviensis, S. viridis, S. xylophagus, S. macrosporeus, S. thermocarboxydus, and S. albogriseolus. The diversity index parameters like Shannon index, reciprocal of Simpson’s index (1/D), and Pielou index of evenness based on ARDRA revealed that streptomycetes community in effluent irrigated field (EIF) was more diverse. DGGE profiles of Streptomyces specific 16S rRNA gene fragments (16S-DGGE) amplified directly from soil samples were highly similar in both soils.     

Investigation of the biotransformation of pentachlorophenol and pulp paper mill effluent decolorisation by the bacterial strains in a mixed culture

Mixed culture of two bacterial strains Bacillus sp. and Serratia marcescens showed potential pentachlorophenol (PCP) degradation and decolorisation of pulp paper mill effluent. The physico-chemical quality of pulp paper mill effluent has been analyzed after 168 h incubation period degraded by mixed culture. The study revealed that it has decreased high load of BOD, COD, TS, TDS, TSS, sulphate, phosphate, total nitrogen, total phenols, metals and different salts (i.e. chloride, sodium, nitrate, potassium) at 168 h incubation period. PCP degradation in pulp paper mill effluent was confirmed by HPLC analysis. Mixed culture was found to degrade PCP up to (94%) present in pulp paper mill effluent with 1% glucose and 0.5% peptone (w/v) at 30 ± 1 °C, pH 8.0 ± 0.2 at 120 rpm in 168 h incubation period. The simultaneous release of chloride ion up to 1200 mg/l at 168 h emphasized the bacterial dechlorination in the medium. The pulp paper mill effluent degradation was also supported by decline in pH, AOX (absorbable organic halides), color, D.O., BOD, COD and PCP. The analysis of pulp paper mill effluent degradation products by GC–MS analysis revealed the formation of low molecular weight compound like 2-chlorophenol (RT = 3.8 min) and tetrachlorohydroquinone (RT = 11.86 min) from PCP extracted degraded sample. Further, mixed culture may be used for bioremediation of PCP containing pulp paper mill waste in the environment.     

Colour removal from bagasse-based pulp mill effluent using a white rot fungus

Colour removal of pulp plant effluent was studied using white rot fungus, Trametes (Coriolus) versicolor. The batch experiments were carried out using fungus in the form of mycelial pellets. In the present investigation, the effect of pH, concentrations of glucose (substrate), initial effluent colour and ammonium chloride (nutrient) on colour removal efficiency were studied. It was found that the maximum colour removal efficiency of 82.5% was obtained with an optimal glucose and ammonium chloride concentrations of 15 g/l and 0.5 g/l respectively at a pH of 4.5 without diluting the effluent.     

Characterization of a fungal strain isolated from a polyphenol polluted site

A group of fungal strains were isolated from a polyphenol polluted soil, taken from an olive oil processing plant in Attica, Greece. The fungi were tested for their ability to decolorize a polyaromatic dye Poly R-478, which was used as a model compound to test their ligninolytic activities. The strain K1.1 decolorized efficiently the dye on agar plates and was further studied. PCR amplification of the internal transcribed spacer (ITS) region of the ribosomal RNA genes from the genomic DNA isolated from mycelium grown in liquid culture resulted in amplified fragments. Via BLASTN search, the length of a 773 base pairs was identified as the basidiomycetes Coprinellus xanthothrix. The growth rates and the tolerance of the fungus were compared on solid media, containing four different concentrations of pentachlorophenol. Extracellular enzyme activities (lignin peroxidase, manganese peroxidase and laccase) were determined in defined liquid medium. The isolate expressed laccase and manganese peroxidase but not lignin peroxidase. The removal of the dye was also estimated in liquid medium. The fungus showed biosorption and biotransformation as removal mechanisms.     

Reaction of estuarine ecosystems to effluent from pulp and paper industry

A major problem associated with the discharge of pulp or paper-mill effluent into shallow estuarine ecosystems, such as the upper L'Etang in Canada or the Don in Scotland, is the development of anoxic conditions in water column and sediment. The effluent modifies the balance, within the ecosystem, between the supply of, and demand for, oxygen. The lignosulphonate component of pulpmill effluent blocks the transmission of light energy to photosynthetic green plants. The oxygen demand is increased by the microbial degration of the additional organic matter in water and sediment. The addition of cellulosic pulp fibre to the sediment also results in an increase in the concentration of their metabolic by-product, sulphide. The oxidation of sulphide exerts an additional oxygen demand on the system, which is an important factor in the development and maintenance of anoxic conditions. It is suggested that temperature is an important factor in controlling the release of sulphide from the sediment into the overlying water and in modifying the toxicity of sulphide towards the bacterial populations and, therefore, the capacity of the ecosystem to cope with the effluent.     

Bioremediation of colour of methyl violet and phenol from a dye-industry waste effluent using Pseudomonas spp. isolated from factory soil

Methyl violet, a basic dye, is manufactured using phenol and dimethylaniline as the raw materials. It is, therefore likely that the waste effluent arising from such units may contain the dye and unused chemicals. Since such pollutants may be toxic, their removal becomes necessary. The studies were therefore aimed at their bioremediation using microbial species.
Four species of Pseudomonas , namely Ps. alcaligenes, Ps. mendocina, Ps. putida biovar B and Ps. stutzeri , were isolated from cattle dung enrichments and the soil samples in the premises of the factory manufacturing methyl violet, nearby Pune.
All the four species of Pseudomonas were able to remove phenol and methyl violet with simultaneous reduction in chemical oxygen demand (COD), total organic carbon (TOC) and ammoniacal nitrogen from the waste effluent and the removal was found to be optimum at the original alkaline pH (range 7.45–10.6) of the waste effluent, at ambient temperature (28 ± 2°C under aerated culture condition, at inoculum density of 1.5 × 10⁸ cells ml⁻¹ of the waste effluent and incubation period of 24–48 h. Thus, Pseudomonas spp. isolated from the environment of the dye industry itself and thus naturally adapted could be used for bioremediation of these pollutants.     

Biodegradation of chlorpyrifos by bacterial consortium isolated from agriculture soil

Organophosphorous pesticides are widely used in agriculture to control major insect pests. Chlorpyrifos is one of the major organophosphorous pesticides which is used to control insects including termites, beetles. The widespread use of these pesticides is hazardous to the environment and also toxic to mammals, thus it is essential to remove the same from the environment. From the chlorpyrifos contaminated soil nine morphologically different bacterial strains, one actinomycete and two fungal strains were isolated. Among those isolates four bacterial strains which were more efficient were developed as consortium. The four bacterial isolates namely Pseudomonas putida (NII 1117), Klebsiella sp., (NII 1118), Pseudomonas stutzeri (NII 1119), Pseudomonas aeruginosa (NII 1120) present in the consortia were identified on the basis of 16S rDNA analysis. The intracellular fractions of the consortium exhibited more organophosphorus hydrolase activity (0.171 ± 0.003 U/mL/min). The degradation studies were carried out at neutral pH and temperature 37°C with chlorpyrifos concentration 500 mg L⁻¹. LC-mass spectral analysis showed the presence of metabolites chlopyrifos-oxon and Diethylphosphorothioate. These results highlight an important potential use of this consortium for the cleanup of chlorpyrifos contaminated pesticide waste in the environment.     

Identification of bacteria contaminating pulp and a paper machine in a Canadian paper mill

Over 100 bacteria from pulp and slime samples in a Canadian paper mill were identified by partial sequencing of their 16S rDNAs. Seventy-one percent of the isolates could be assigned to a bacterial genus with a high level of confidence. Another 12% exhibited at least 95% similarity within their 16S rDNA sequence with unidentified organisms that originate from warm or wet environments. Pseudomonas, Bacillus, and Pseudoxanthomonas isolates were represented at a relatively high proportion in both pulp and slime samples. This is the first time that Pseudoxanthomonas strains have been isolated from pulp and slime samples on a paper machine. Electronic Publication     

Genetic and functional diversity of Bacillus strains in the soils long-term irrigated with paper and pulp mill effluent

The genetic and functional diversity of Bacillus and Bacillus-derived genera was analyzed in soil samples collected from three different fields near Century Paper Mill, Lal Kuan, Uttarakhand, India. Two had been subjected to concentrated and diluted effluent irrigation for the past 25 years and were designated as a concentrated effluent irrigated field (CEIF) and a dilute effluent irrigated field (DEIF), respectively. The field irrigated with fresh water was designated as a water irrigated field (WIF). Increase in pH, Na and Zn content and decrease in Fe content was observed due to effluent irrigation. The population count of Bacillus and Bacillus-derived was maximum in DEIF followed by WIF and CEIF. Variations in plant growth-promoting traits and extracellular enzymes were observed among the isolates from the three different field soils. Based on the amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes, all the selected 104 isolates were clustered into 14 groups. The sequencing of the representative isolates revealed that the majority belonged to the genus Bacillus, while three isolates belonged to Paenibacillus, Lysinibacillus and Orthinibacillus. There were a few species like Orthinibacillus contaminans, B. oleronius, B. safensis, B. methylotrophicus, B. stratosphericus, B. aryabhattai, B. asahii and B. bataviensis that were prevalent only in DEIF and CEIF but not in WIF field soil. The diversity index parameters like the Shannon Index, indices of species richness and species evenness based on biochemical profiling and ARDRA profiling revealed that Bacillaceae members in the fresh water irrigated field were diverse.     

Colour remediation of pulp mill effluent using purified fungal cellobiose dehydrogenase: reaction optimisation and mechanism of degradation

Cellobiose dehydrogenase purified from two different fungal sources was assessed for its ability to remove and/or reduce colour from pulp mill bleach plant effluent. Cellobiose dehydrogenase purified from Phanerochaete chrysosporium was shown to prefer acidic conditions and was consequently used to treat the acid effluent stream discharged from a pulp mill bleach plant, while an analogous enzyme originating from Humicola insolens preferred alkaline conditions, and was applied to the effluent discharged from the caustic sewer of the bleach plant. Both enzyme preparations were able to remove colour from their respective effluent sources to a comparable extent. Up to 50% of the effluent colour was removed within 4 days when treated under optimised conditions. Furthermore, it was also shown that this enzymatic approach was effective at removing colour generated by both softwood and hardwood resources. Mechanistically, it was shown that colour was removed from all molecular weight fractions, and the higher molecular weight material (>300 kDa) was concurrently preferentially degraded. Cellobiose dehydrogenase treatment of effluent did not target phenolic, stilbene, or alpha-carbonyl structures, but did affect the quinone content. Further investigations using model compounds confirmed these results, and subsequently showed that only the para-quinones with low substitution were reduced with cellobiose dehydrogenase.