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.     

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.     

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.     

Genotoxic activity of pulp mill effluent in Salmonella and Saccharomyces cerevisiae assays

An XAD-2 resin concentrate of chlorination-stage pulp mill effluent was found to induce mutations in Salmonella typhimurium strains TA1535, TA100 and TA98 but not in strains TA1537 or TA1538. The presence of either S9 mix, S9 mix without cofactors, or heat-inactivated S9 mix, reduced the mutagenic effects. Dose-related increases in gene conversion, mitotic recombination and aberrant colony formation in Saccharomyces cerevisiae strain D7 also were found.     

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.     

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。     

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.     

Effect of effluent from a nitrogen fertilizer factory and a pulp mill on the distribution and abundance of Aeromonas hydrophila in Albemarle Sound, North Carolina.

The density of Aeromonas hydrophila, standard count bacteria, fecal coliform bacteria, and 18 physical and chemical parameters were measured simultaneously at six sites for 12 months in Albemarle Sound, N.C. One site was above and two sites were below the discharge plume of a Kraft pulping process paper mill. The fourth site was above and the remaining two sites were below the discharge point of a nitrogen fertilizer factory. The impact of the pulp mill on water quality was acute, whereas that of the nitrogen fertilizer factory was chronic and much more subtle. Diffusion chamber studies indicated that A. hydrophila survival is increased by pulp mill effluent and decreased by nitrogen fertilizer factory effluent. From correlation and regression analysis, A. hydrophila was found to be directly affected by phytoplankton density and, thus, indirectly by concentrations of phosphate, nitrate, and total organic carbon. These two point sources are suspect as indirect causes of red-sore disease epizootics, a disease of fish caused by A. hydrophila.     

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.     

Chitin: a potential new alternative nitrogen source for the tertiary,algal-based treatment of pulp and paper mill wastewater

Every day, pulp and paper mills in the USA discharge millions of liters of wastewater. Primary and secondary treatment of this wastewater often enriches it with phosphorus, resulting in uncontrolled eutrophication of receiving water bodies. A new method of tertiary wastewater treatment uses controlled growth of algae in a photobioreactor to sequester phosphorus into algal biomass, which is then harvested. This typically requires addition of a nitrogen fertilizer (nitrate, ammonium, or urea) to the water. We show on the laboratory scale that chitin can be used as an alternative source of nitrogen for the tertiary treatment of pulp mill wastewater using algae. We demonstrate that phosphorus can be efficiently removed from pulp wastewater using algae and chitin. Furthermore, phosphorus removal with chitin did not result in an increase in dissolved nitrogen in the wastewater because it is insoluble, unlike conventional nitrogen fertilizers. Despite its insolubility, it has recently been found that many diverse algae and cyanobacteria can use it as a source of nitrogen. Chitin has many advantages over conventional nitrogen fertilizers for use in wastewater treatment technologies. It is the second-most abundant natural polymer and is a waste product of the shellfish industry. Chitin is sustainable, inexpensive, and carbon neutral. Thus, chitin improves the sustainability and carbon footprints associated with water treatment, while the production of commercially attractive algal biomass helps to offset costs associated with the water treatment system itself.     

Laccase induction in fungi and laccase/N-OH mediator systems applied in paper mill effluent

There has been increasing interest in extracellular enzymes from white rot fungi, such as lignin and manganese peroxidases, and laccases, due to their potential to degrade both highly toxic phenolic compounds and lignin. The optimum cultivation conditions for laccase production in semi-solid and liquid medium by Trametes versicolor, Trametes villosa, Lentinula edodes and Botrytis cinerea and the effects of laccase mediator system in E1 effluent were studied. The higher laccase activity (12756 U) was obtained in a liquid culture of T. versicolor in the presence of 1 mM of 2,5-xylidine and 0.4 mM copper salt as inducers. The effluent biotreatments were not efficient in decolorization with any fungal laccases studied. Maximum phenol reduction was approximately 23% in the absence of mediators from T. versicolor. The presence of 1-hydroxybenzotriazole did not increase phenol reduction. However, acetohydroxamic acid, which was not degraded by laccase, acted very efficiently on E1 effluent, reducing 70% and 73% of the total phenol and total organic carbon, respectively. Therefore, acetohydroxamic acid could be applied as a mediator for laccase bioremediation in E1 effluent.     

Effect of effluent from a nitrogen fertilizer factory and a pulp mill on the distribution and abundance of Aeromonas hydrophila in Albemarle Sound, North Carolina

The density of Aeromonas hydrophila, standard count bacteria, fecal coliform bacteria, and 18 physical and chemical parameters were measured simultaneously at six sites for 12 months in Albemarle Sound, N.C. One site was above and two sites were below the discharge plume of a Kraft pulping process paper mill. The fourth site was above and the remaining two sites were below the discharge point of a nitrogen fertilizer factory. The impact of the pulp mill on water quality was acute, whereas that of the nitrogen fertilizer factory was chronic and much more subtle. Diffusion chamber studies indicated that A. hydrophila survival is increased by pulp mill effluent and decreased by nitrogen fertilizer factory effluent. From correlation and regression analysis, A. hydrophila was found to be directly affected by phytoplankton density and, thus, indirectly by concentrations of phosphate, nitrate, and total organic carbon. These two point sources are suspect as indirect causes of red-sore disease epizootics, a disease of fish caused by A. hydrophila.     

On-line enzyme activity determination using the stopped-flow technique: application to laccase activity in pulp mill waste-water treatment

An automated system for on-line measurement of enzyme activity is proposed. The system uses a flow injection manifold in the stopped-flow mode to measure initial reaction rates. The time during which the flow is halted is selected in such a way as to optimise the enzyme/substrate ratio for the correct determination of activity values. The proposed system was used to determine the activity of laccase produced by the fungus Trametes versicolor immobilised on nylon in a fixed-bed reactor used for treating pulp mill waste water. Received: 17 February 1997 / Received revision: 23 April 1997 / Accepted: 27 April 1997     

Screening and identification of microbial strains for removal of colour and adsorbable organic halogens in pulp and paper mill effluent

Eight fungal and three bacterial isolates collected from decomposed wood, sediment core and effluent of pulp and paper mill were evaluated for their ability to decolourize kraft pulp bleached effluents. Decolourization potency of Paecilomyces sp. (F₃) was maximal (67%) on day 1 followed by F₅ (Phoma sp.) and F₇ (Paecilomyces varioti). Among the various carbon sources used, Paecilomyces sp. (F₃) reduced more than 80% colour and lignin in the presence of minimal salt medium and dextrose (0.2%, w/v), and there was an increase in biomass from 8.1 mg/ml initially to 12.8 mg/ml during that period. In the batch reactor one of the three bacteria, Pseudomonas aeruginosa, removed 48% colour from the effluent after 1 day followed by Acinetobacter calcoaceticus (39%) and Klebsiella pneumoniae (25%). In a two stage sequential bioreactor strain F₃ was able to reduce 68% colour and P. aeruginosa 34% in 1 day. However, when fungal treated effluent was subsequently treated by P. aeruginosa 82% colour was reduced. The reduction of adsorbable organic halogens (AOX) in effluent was determined by F₃ strain, however, bacterial strain PCP2 increased the content initially on day 1, which was readily degraded after 3 days by both fungus and bacterium in the sequential bioreactor.     

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.     

Evaluation of tertiary treatment by fungi,enzymatic and photo-Fenton oxidation on the removal of phenols from a kraft pulp mill effluent: a comparative study

Pulp and paper mills generate pollutants associated to their effluents depending upon the type of process, type of the wood materials, process technology applied, management practices, internal recirculation of the effluent for recovery, the amount of water used in the industrial process and type of secondary treatment. This study is the first that reports a simultaneous evaluation of the effects of tertiary treatments by fungi (Rhizopus oryzae and Pleurotus sajor caju), by enzyme (laccase) and by an oxidation process (photo-Fenton) on individual phenols (vanillin, guaiacol, phloroglucinol, vanillic acid and syringic acid) of a Eucalyptus globulus bleached kraft pulp and paper mill final effluent after secondary treatment (BKPME). The tertiary treatments were applied on BKPME samples and in BKPME samples supplemented with extra concentration of each phenol. Tertiary treatments by Rhizopus oryzae and photo-Fenton oxidation were able of complete removal (100%) of phenols on BKPME samples whereas P. sajor caju and laccase were able of 60–85% removal. On BKPME samples with added concentration of each phenol, photo-Fenton was the only treatment capable of total phenols removal (100%), which suggests a great potential for its application.     

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.     

Co-occurrence of in-stream nitrogen fixation and denitrification across a nitrogen gradient in a western U.S. watershed

It is frequently assumed that nitrogen (N₂) fixation and denitrification do not co-occur in streams because each process should be favored under different concentrations of dissolved inorganic nitrogen (DIN), and therefore these processes are rarely quantified together. We asked if these processes could co-exist by conducting a spatial survey of N₂ fixation using acetylene reduction and denitrification using acetylene block [with and without amendments of carbon (C) as glucose and nitrogen (N) as nitrate]. Rates were measured on rocks and sediment in 8 southeastern Idaho streams encompassing a DIN gradient of 26–615 µg L^?1. Sampling at each site was repeated in summer 2015 and 2016. We found that both denitrification and N₂ fixation occurred across the gradient of DIN concentrations, with N₂ fixation occurring primarily on rocks and denitrification occurring in sediment. N₂ fixation rates on rocks significantly decreased 100× across the DIN gradient in 1 year of the study, and amended (with N and C) denitrification rates increased 10× across the DIN gradient in both years. Multiple linear regression and partial least squares models with environmental characteristics measured at the scale of entire stream reaches showed that C and phosphorus were positive predictors of amended and unamended denitrification rates, but no significant model could explain N₂ fixation rates across all streams and years. This, coupled with the observation that detectable rates of N₂ fixation occurred primarily on rocks and denitrification occurred primarily on sediment, suggests that microhabitat scale factors may better predict the co-occurrence of these processes within stream reaches. Overlooking the potential co-occurrence of N₂ fixation and denitrification in stream ecosystems will impede understanding by oversimplifying the contribution of each process to the N cycle.     

Energy decision making in a pulp and paper mill: selection of LCA system boundary

Background, aim, and scope  

North American pulp and paper mills are facing tremendous challenges, which may necessitate major mill modernizations. An example is process modification to reduce dependency on purchased power, which is an expensive resource. Such modifications may have environmental implications at the mills’ sites, on their product life cycle, and on other interconnected systems, and therefore, systematic tools such as life cycle assessment (LCA) need to be applied. Different LCA system boundary approaches can be used for such process design applications, and these approaches need to be compared to determine their respective benefits and limitations in this context. This study compares setting the system boundary according to a cradle-to-gate approach [attributional LCA (ALCA)] and a system expansion [consequential LCA (CLCA)] approach using a case study, which deals with implementing cogeneration and increased de-inked pulp production at an integrated newsprint mill.     

Effects of pulp and paper mill effluents on estuarine and marine ecosystems in Canada: a review

This review examines the impact of pulp and paper mill effluents by comparing effects from Canada's east and west coasts at a time when revisions to the federal Fisheries Act (Pulp and Paper Effluent Regulations) are being finalized. Pulp and paper mill effluents from Canadian coastal mills were usually acutely toxic at source, and in many cases had marked deleterious effects on receiving waters due to toxicity, high biochemical oxygen demand (BOD), and total suspended solids (TSS) loadings. Extreme reductions in ambient dissolved oxygen, impacts on benthic and intertidal organisms, changes in water colour and primary productivity, have been demonstrated over the years and continue to cause environmental damage. Contamination of biota by a wide range of chlorinated organic compounds has been more recently the focus of investigations.While sublethal effects of lowered dissolved oxygen levels and suspended solids on the water column and bottom communities are well known, the potential effects of major organochlorine contamination of water (measured as Adsorbable Organic Halogens=AOX), sediments, and biota are not fully understood, especially under natural and perturbated conditions. The findings of recent North American and Scandinavian studies which describe liver enzyme activation, histological damage, reproductive and population level changes in fish, are a major concern as they are a sign of ecosystem stress and pathology.The environmental effects described herein are long-term impacts which will not respond quickly to changes in pollutant loading. Integrated site-specific assessments need to be undertaken to document ecosystem response to process and treatment improvements at mill sites. Current biomonitoring techniques including measures of population structure and ecosystem function are needed in addition to sensitive biochemical indicators of contaminant exposure.