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In recent years, whole-cell biosensors (WCBs) have emerged as a potent approach for environmental monitoring and on-site analyte detection. These biosensors harness the biological apparatus of microorganisms to identify specific analytes, offering advantages in sensitivity, specificity, and real-time monitoring capabilities. A critical hurdle in biosensor development lies in ensuring the robust attachment of cells to surfaces, a crucial step for practical utility. In this study, we present a comprehensive approach to tackle this challenge via engineering Escherichia coli cells for immobilization on paper through the Curli biofilm pathway. Furthermore, incorporating a cellulose-binding peptide domain to the CsgA biofilm protein enhances cell adhesion to paper surfaces, consequently boosting biosensor efficacy. To demonstrate the versatility of this platform, we developed a WCB for copper, optimized to exhibit a discernible response, even with the naked eye. To confirm its suitability for practical field use, we characterized our copper sensor under various environmental conditions-temperature, salinity, and pH-to mimic real-world scenarios. The biosensor-equipped paper discs can be freeze-dried for deployment in on-site applications, providing a practical method for long-term storage without loss of sensitivity paper discs demonstrate sustained functionality and viability even after months of storage with 5 μM limit of detection for copper with visible-to-naked-eye signal levels. Biofilm-mediated surface attachment and analyte sensing can be independently engineered, allowing for flexible utilization of this platform as required. With the implementation of copper sensing as a proof-of-concept study, we underscore the potential of WCBs as a promising avenue for the on-site detection of a multitude of analytes., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Urartu Ozgur Safak Seker reports financial support was provided by "The Scientific and Technological Research Council of Turkey". If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: The main objective of this study is the evaluation of RPPW anaerobic digestion feasibility at laboratory scale under Mesophilic condition. The experiment is conducted using a two-stage biofilm digester of 5 L capacity with mobile support material., Results: Anaerobic treatment of wastewater from recycled pulp and paper industry in Morocco was tested using a laboratory-scale anaerobic biofilm digester that operated under mesophilic conditions over a 70-day. Chemical oxygen demand (COD) efficiency, volatile and total solid (VS, TS) elimination of the substrate during the process were: 78%, 52% and 48% respectively. The system was stable throughout its operating cycle with an optimum pH (7.24), alkalinity (1750 mg CaCO 3 /L) and a volatile fatty acid value (760 mg/L). The experimental daily biogas production measured reaches a value of 5 L/day with a composition of 71% methane, 27.6% carbon dioxide, 0.2 oxygen and 7713 ppm of the H 2 S. The study results show that the anaerobic biofilm reactor is a suitable technique for recycled pulp and paper wastewater (RPPW) treatment. The reactor shows high performances in terms of process stability, removal efficiency (> 70%) and biogas production., Conclusion: Anaerobic digestion is an efficient waste treatment technology that uses natural anaerobic decomposition to reduce the volume of waste while producing biogas. However, research is needed to strengthen microbial metabolism, biochemistry and the functioning of the rector to improve biogas production. The RPPW AD experiment with biofilm digester technology was stable throughout the operation period. The digester knows an overloaded in the last phase of the experiment which leads to an inhibition of biogas production.

Secondary effluent from paper mill was characterized by poor biodegradability and containing recalcitrant compounds. In this study, four bioreactors, including a sequencing batch biofilm reactor (SBBR), a stirred-tank reactor (STR) and two submerged aeration reactors (SAR) were used to treat secondary effluent from a recycled paper mill respectively. The results indicated that chemical oxygen demand (COD) was increased by SAR2 treatment and COD removal efficiency for SBBR, SAR1 and STR was 39.7%, 15.7% and 30.9% respectively. It is suggested that recalcitrant compounds were removed by SBBR, SAR1 and STR respectively. Total nitrogen (TN) and total phosphorus (TP) of wastewater were increased by treatments of each bioreactor, which suggested that endogenous respiration of biomass occurred during the treatment. Microbial analysis of sludge from different bioreactors suggested that the removal of recalcitrant compounds in SBBR and STR might be related to the presence of unique microorganisms in each reactor., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Biofilm formation is a major cause of reduced paper quality and increased down time during paper manufacturing. This study uses Illumina next-generation sequencing to identify the microbial populations causing quality issues due to their presence in biofilms and slimes. The paper defects investigated contained traces of the films and/or slime of mainly two genera, Tepidimonas and Chryseobacterium. The Tepidimonas spp. found contributed on average 68% to the total bacterial population. Both genera have been described previously to be associated with biofilms in paper mills. There was indication that Tepidimonas spp. were present as compact biofilm in the head box of one paper machine and was filtered out by the paper web during production. On the other hand Tepidimonas spp. were also present to a large extent in the press and white waters of two nonproblematic paper machines. Therefore, the mere presence of a known biofilm producer alone is not sufficient to cause slimes and therefore paper defects and other critical factors are additionally at play. For instance, we identified Acidovorax sp., which is an early colonizer of paper machines, exhibiting the ability to form extracellular DNA matrices for attachment and biofilm formation., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

A stainless steel paper-embedded biofilm reactor (PEBR) was developed for Candida spp. growth, permitting confluent distribution of nutrients by capillary diffusion through ordinary laboratory filter paper. Antibiogram disks were distributed along the filter paper rim, and the PEBR received 0.1 or 0.01 % crystal violet (CV) at 200 μL min(-1) and at 37 °C, for 48 h. CV was recovered from the disks and measured at 540 nm. Candida albicans SC5314 cells were applied onto antibiogram disks. The bioreactor was assembled, and YEPD broth was admitted (200 μL min(-1)) at 37 °C, for 72 h. Biofilm growth was estimated via the MTT reduction test. Controls were disks that received the same treatments, except for the fungus. The PEBR was considered high-throughput table, low-cost, and feasible to grow C. albicans biofilms.

There are wide spread bacterial contamination issues on various paper products, such as paper towels hanging in sink splash zones or those used to clean surfaces, filter papers used in water and air purifying systems, and wrappings used in the food industry; such contamination may lead to the potential spread of bacteria and consequent severe health concerns. In this study, selenium nanoparticles were coated on normal paper towel surfaces through a quick precipitation method, introducing antibacterial properties to the paper towels in a healthy way. Their effectiveness at preventing biofilm formation was tested in bacterial assays involving Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus epidermidis. The results showed significant and continuous bacteria inhibition with about a 90% reduction from 24 to 72 hours for gram-positive bacteria including S. aureus and S. epidermidis. The selenium coated paper towels also showed significant inhibition of gram-negative bacteria like P. aeruginosa and E. coli growth at about 57% and 84%, respectively, after 72 hours of treatment. Therefore, this study established a promising selenium-based antibacterial strategy to prevent bacterial growth on paper products, which may lead to the avoidance of bacteria spreading and consequent severe health concerns.

The present clinical study aimed to investigate if there are differences in microbiological outcomes dependent on the subgingival biofilm collection method. Subgingival biofilm samples were collected from the four deepest pockets (>5 mm) of 17 patients with aggressive periodontitis (AgP) and 33 patients with chronic periodontitis (CP), first by paper point and thereafter by curette. Samples obtained with the same method were pooled together from each patient and forwarded for molecular microbiological analysis by a commercially available assay (IAI Pado Test 4.5) that estimates total bacterial load and levels of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola and Aggregatibacter actinomycetemcomitans. Data analysis included frequency of detection, quantification and correlation of detection levels between the two sampling methods. P. gingivalis, T. forsythia and T. denticola were detected in >90% of the samples, and their detection levels exhibited a strong correlation between sampling methods. The detection consistency of A. actinomycetemcomitans was 56% between the two sampling methods. A. actinomycetemcomitans was more readily detected by paper point compared with curette collection with a stronger correlation between the two methods in AgP. Subgingival biofilm sampling by curette or paper point does not yield differences in the detection of the three 'red complex' species. However, A. actinomycetemcomitans was more consistently detected by means of paper point collection, which can be crucial in the decision to administer antibiotics as an adjunctive periodontal treatment., (© 2013 APMIS. Published by John Wiley & Sons Ltd.)

Biofilms can cause severe problems in industrial paper mills, particularly of economic and technological types (clogging of filters, sheet breaks or holes in the paper, machine breakdowns, etc.). We present here some promising results on the use of essential oil compounds to control these biofilms. Biofilms were grown on stainless-steel coupons with a microbial white water consortium sampled from an industrial paper mill. Five essential oil compounds were screened initially in the laboratory in terms of their antimicrobial activity against planktonic cells and biofilms. The three most active compounds were selected and then tested in different combinations. The combination finally selected was tested at the pilot scale to confirm its efficiency under realistic conditions. All the compounds tested were as active against biofilms as they were against planktonic cells. The most active compounds were thymol, carvacrol, and eugenol, and the most efficient combination was thymol-carvacrol. At a pilot scale, with six injections a day, 10 mM carvacrol alone prevented biocontamination for at least 10 days, and a 1 mM thymol-carvacrol combination enabled a 67 % reduction in biofilm dry matter after 11 days. The use of green antimicrobials could constitute a very promising alternative or supplement to the treatments currently applied to limit biofilm formation in the environment of paper mill machines.

Phosphate is an essential nutrient for heterotrophic bacteria, affecting bacterioplankton in aquatic ecosystems and bacteria in biofilms. However, the influence of phosphate limitation on bacterial competition and biofilm development in multispecies populations has received limited attention in existing studies. To address this issue, we isolated 13 adhesive bacteria from paper machine aggregates. Intergeneric inhibition of Pseudomonas aeruginosa WW5 by Serratia marcescens WW4 was identified under phosphate-limited conditions, but not in Luria-Bertani medium or M9 minimal medium. The viable numbers of the pure S. marcescens WW4 culture decreased over 3 days in the phosphate-limited medium; however, the mortality of S. marcescens WW4 was significantly reduced when it was co-cultured with P. aeruginosa WW5, which appeared to sustain the S. marcescens WW4 biofilm. In contrast, viable P. aeruginosa WW5 cells immediately declined in the phosphate-limited co-culture. To identify the genetic/inhibitory element(s) involved in this process, we inserted a mini-Tn5 mutant of S. marcescens WW4 that lacked inhibitory effect. The results showed that an endonuclease bacteriocin was involved in this intergeneric inhibition by S. marcescens WW4 under phosphate limitation. In conclusion, this study highlights the importance of nutrient limitation in bacterial interactions and provides a strong candidate gene for future functional characterisation., (© 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Bacterial infections are commonly found on paper towels and other paper products, leading to the potential spread of bacteria and consequent health concerns. The objective of this in vitro study was to introduce antibacterial properties to standard paper towel surfaces by coating them with selenium nanoparticles. Scanning electron microscopy was used to measure the size and distribution of the selenium coatings on the paper towels. Atomic force microscopy was used to measure the surface roughness of paper towels before and after they were coated with selenium nanoparticles. The amount of selenium precipitated on the paper towels was measured by atomic absorption spectroscopy. In vitro bacterial studies with Staphylococcus aureus were conducted to assess the effectiveness of the selenium coating at inhibiting bacterial growth. Results showed that the selenium nanoparticles coated on the paper towel surface were well distributed with semispherical geometries about 50 nm in diameter. Most importantly, the selenium nanoparticle-coated paper towels inhibited S. aureus growth by 90% after 24 and 72 hours compared with the uncoated paper towels. Thus, the study showed that nanoparticle selenium-coated paper towels may lead to an increased eradication of bacteria in a wider range of clinical environments and in the food industry, thus improving human health.

The use of enzymes has a high potential in the pulp and paper industry to improve the economics of the paper production process and to achieve, at the same time, a reduced environmental impact. Specific enzymes contribute to reduce the amount of chemicals and energy required for the modification of fibers and helps to prevent the formation or development of biofilms. This review is aimed at presenting the latest progresses made in the application of enzymes as refining aids and biofilm control agents.

Polyhydroxyalkanoate (PHA) biosynthesis in paper mill wastewater treated by a Moving Bed Biofilm Reactor (MBBR) was evaluated. A MBBR was operated during 300 d. The increasing effect of the Organic Load Rate (OLR) from 0.13 kg BOD(5)/m(3)·d to 2.99 kg BOD(5)/m(3)·d and the influence of two relationship of BOD(5:) N: P (100: 5: 1 and 100: 1: 0.3) on the PHA biosynthesis were evaluated. With an OLR of 0.13 kg BOD(5)/m(3)·d, the maximum organic matter removal measure as Biochemical Oxygen Demand (BOD(5)) was 98.7% for a BOD(5:) N: P relationship of 100: 5: 1. Meanwhile for BOD(5): N: P relationship of 100: 1: 0.3, the maximum efficiency was 87.2% (OLR: 2.99 kg BOD(5)/m(3)·d). The behaviour of the Chemical Oxygen Demand (COD) and total phenolic compound removal efficiencies were below 65.0% and 41.0%, respectively. PHA biosynthesis was measured as a percentage of cells that accumulate PHA, where the maximum percentage was 85.1% and 78.7% when MBBR was operated under a BOD(5): N: P relationship of 100: 5: 1 and 100: 1: 0.3, respectively. Finally, the PHA yields in this study were estimated to range between 0.11 to 0.72 mg PHA/mg VSS and 0.06 to 0.15 mg PHA/mg COD.

This study aimed to characterize biofilms from the paper industry and evaluate the effectiveness of enzymatic treatments in reducing them. The extracellular polymeric substances (EPS) extracted from six industrial biofilms were studied. EPS were mainly proteins, the protein to polysaccharide ratio ranging from 1.3 to 8.6 depending on where the sampling point was situated in the paper making process. Eight hydrolytic enzymes were screened on a 24-h multi-species biofilm. The enzymes were tested at various concentrations and contact durations. Glycosidases and lipases were inefficient or only slightly efficient for biofilm reduction, while proteases were more efficient: after treatment for 24 h with pepsin, Alcalase® or Savinase®, the removal exceeded 80%. Savinase® appeared to be the most adequate for industrial conditions and was tested on an industrial biofilm sample. This enzyme led to a significant release of proteins from the EPS matrix, indicating its potential efficiency on an industrial scale.

The antifouling potential of electric polarization combined and not combined with biocides was studied in nonsaline warm water with high organic content. Deinococcus geothermalis is a bacterium known for forming colored biofilms in paper machines and for its persistence against cleaning and chemical treatments. When D. geothermalis biofilms grown for 24 h in simulated paper machine water were exposed to cathodic or cathodically weighted pulsed polarization at least 60% (P < 0.05) of the biofilms were removed from stainless steel (AISI 316L). Biofilm removal by 25 ppm (effective substances 5-25 ppm) of oxidizing biocides (bromochloro-5,5-dimethylhydantoin, 2,2-dibromo-2-cyanoacetamide, peracetic acid) increased to 70% when combined with cathodically weighted pulsed polarization. Using a novel instrument that allows real-time detection of reactive oxygen species (ROS) we showed that the polarization program effective in antifouling generated ROS in a pulsed manner on the steel surface. We thus suggest that the observed added value of oxidative biocides combined with polarization depended on ROS. This suggestion was supported by the finding that a reductive biocide, methylene bisthiocyanate, counteracted the antifouling effect of polarization.

Microbiological control programmes at industrial level should aim at reducing both the detrimental effects of microorganisms on the process and the environmental impact associated to the use of biocides as microbiological control products. To achieve this target, new efficient and environmentally friendly products are required. In this paper, 17 non-specific, commercial enzymatic mixtures were tested to assess their efficacy for biofilm prevention and control at laboratory and pilot plant scale. Pectin methylesterase, an enzyme found in the formulation of two of the mixtures tested, was identified as an active compound able to reduce biofilm formation by 71% compared to control tests.

Biofilms cause runnability problems in paper machines and are therefore controlled with biocides. Peracetic acid is usually effective in preventing bulky biofilms. This study investigated the microbiological status of a paper machine where low concentrations (≤ 15 ppm active ingredient) of peracetic acid had been used for several years. The paper machine contained a low amount of biofilms. Biofilm-forming bacteria from this environment were isolated and characterized by 16S rRNA gene sequencing, whole-cell fatty acid analysis, biochemical tests, and DNA fingerprinting. Seventy-five percent of the isolates were identified as members of the subclades Sphingomonas trueperi and S. aquatilis, and the others as species of the genera Burkholderia (B. cepacia complex), Methylobacterium, and Rhizobium. Although the isolation media were suitable for the common paper machine biofoulers Deinococcus, Meiothermus, and Pseudoxanthomonas, none of these were found, indicating that peracetic acid had prevented their growth. Spontaneous, irreversible loss of the ability to form biofilm was observed during subculturing of certain isolates of the subclade S. trueperi. The Sphingomonas isolates formed monoculture biofilms that tolerated peracetic acid at concentrations (10 ppm active ingredient) used for antifouling in paper machines. High pH and low conductivity of the process waters favored the peracetic acid tolerance of Sphingomonas sp. biofilms. This appears to be the first report on sphingomonads as biofilm formers in warm water using industries.

Methanol-oxidizing bacteria may play an important role in the development and use of biological treatment systems for the removal of methanol from industrial effluents. Optimization of methanol degradation potential in such systems is contingent on availability of nutrients, such as nitrogen, in the most favorable form and concentration. To that end, this study examined the variation in growth, methanol degradation, and bacterial diversity of two mixed methylotrophic cultures that were provided nitrogen either as ammonium or nitrate and in three different concentrations. Methanol-degrading cultures were enriched from biofilms sampled at a pulp and paper mill and grown in liquid batch culture with methanol as the only carbon source and either ammonium or nitrate as the only added nitrogen source. Results indicate that growth and methanol removal of the mixed cultures increase directly with increased nitrogen, added in either form. However, methanol removal and bacterial diversity, as observed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) methods, were higher when using nitrate as the nitrogen source for enrichment and growth, rather than ammonium. Based on results described here, nitrate may potentially be a better nitrogen source when enriching or working with mixed methylotrophic cultures, and possibly more effective when used as a nutrient addition to biofilters.

A rod-shaped, non-spore-forming, non-motile, aerobic, oxidase and catalase-positive and radiation-resistant bacterium (designated strain K4.1(T)) was isolated from biofilm collected from a Finnish paper mill. The bacterium grew as pale pink colonies on oligotrophic medium at 12 to 50 °C (optimum 37 to 45 °C) and at pH 6 to 10.3. The DNA G+C content of the strain was 66.8 l%. According to 16S rRNA gene sequence analysis, strain K4.1(T) was distantly related to the genus Deinococcus, sharing highest similarity with Deinococcus pimensis (90.0  %). In the phylogenetic tree, strain K4.1(T) formed a separate branch in the vicinity of the genus Deinococcus. The peptidoglycan type was A3β with L-Orn-Gly-Gly and the quinone system was determined to be MK-8. The polar lipid profile of strain K4.1(T) differed markedly from that of the genus Deinococcus. The predominant lipid of strain K4.1(T) was an unknown aminophospholipid and it did not contain the unknown phosphoglycolipid predominant in the polar lipid profiles of deinococci analysed to date. Two of the predominant fatty acids of the strain, 15 : 0 anteiso and 17 : 0 anteiso, were lacking or present in small amounts in species of the genus Deinococcus. Phylogenetic distinctness and significant differences in the polar lipid and fatty acid profiles suggest classification of strain K4.1(T) as a novel genus and species in the family Deinococcaceae, for which we propose the name Deinobacterium chartae gen. nov., sp. nov. The type strain is K4.1(T) (=DSM 21458(T) =HAMBI 2721(T)).

During the process of papermaking by pulp and paper plants, a thick and viscous deposits, termed slime, is quickly formed around the paper machines, which can affect the papermaking process. In this study, we explored the composition of the bacterial biota in slime that developed on shower pipes from 2 machines at a Canadian paper mill. Firstly, the composition was assessed for 12 months by DNA profiling with polymerase chain reaction coupled with denaturing gradient gel electrophoresis. Except for short periods (2-3 months), clustered analyses showed that the bacterial composition of the slime varied substantially over the year, with less than 50% similarity between the denaturing gradient gel electrophoresis profiles. Secondly, the screening of 16S rRNA gene libraries derived from 2 slime samples showed that the most abundant bacteria were related to 6 lineages, including Chloroflexi, candidate division OP10, Clostridiales, Bacillales, Burkholderiales, and the genus Deinococcus. Finally, the proportion of 8 bacterial lineages, such as Deinococcus sp., Meiothermus sp., and Chloroflexi, was determined by the Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization in 2 slime samples. The results showed a high proportion of Chloroflexi, Tepidimonas spp., and Schlegelella spp. in the slime samples.

Biofilm samples collected from inside and outside the press and former sections of paper machines in a Northwestern Ontario paper mill for a period of 2 years were characterized microbiologically and electrochemically. Bacterial community profiling was done using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and selected bacterial isolates were identified using 16S rDNA analysis. The bacterial community showed the presence of Proteobacteria, Firmicutes, and Actinobacteria. Sphingomonas sp. was found to be the most common bacterial species, which showed the highest production of extracellular polymeric substances. Bacteria isolated from biofilms showed better adhesion properties than those from water samples. Cyclic voltammetry and electrochemical impedance spectroscopy studies showed that bacteria isolated from biofilms and feed water collected from inside the machine were more easily oxidized than those from outside, suggesting the need for a more rigorous biofilm abatement strategy for inside paper machines.

A paper mill producing 500,000 ton of graphic paper annually has an on-site wastewater treatment plant that treats 7,240,000 m³ of wastewater per year, mechanically first, then biologically and at last by ozonation. Increased paper production capacity led to higher COD load in the mill effluent while production of higher proportions of brighter products gave worse biodegradability. Therefore the biological capacity of the WWTP needed to be increased and extra measures were necessary to enhance the efficiency of COD reduction. The full scale implementation of one MBBR with a volume of 1,230 m³ was accomplished in 2000 followed by another MBBR of 2,475 m³ in 2002. An ozonation step with a capacity of 75 kg O₃/h was added in 2004 to meet higher COD reduction demands during the production of brighter products and thus keeping the given outflow limits. Adding a moving bed biofilm reactor prior to the existing activated sludge step gives: (i) cost advantages when increasing biological capacity as higher COD volume loads of MBBRs allow smaller reactors than usual for activated sludge plants; (ii) a relief of strain from the activated sludge step by biological degradation in the MBBR; (iii) equalizing of peaks in the COD load and toxic effects before affecting the activated sludge step; (iv) a stable volume sludge index below 100 ml/g in combination with an optimization of the activated sludge step allows good sludge separation--an important condition for further treatment with ozone. Ozonation and subsequent bio-filtration pre-treated waste water provide: (i) reduction of hard COD unobtainable by conventional treatment; (ii) controllable COD reduction in a very wide range and therefore elimination of COD-peaks; (iii) reduction of treatment costs by combination of ozonation and subsequent bio-filtration; (iv) decrease of the color in the ozonated wastewater. The MBBR step proved very simple to operate as part of the biological treatment. Excellent control of the COD-removal rate in the ozone step allowed for economical usage and therefore acceptable operation costs in relation to the paper production.

Formation of biofilms causes severe problems in paper machines, and hence financial costs. It would be preferable to prevent attachment of the primary-colonizing bacteria than to control the growth of secondary communities, which are sheltered by exopolysaccharide slime layers. We have therefore investigated the early succession of paper-machine biofilms by incubating stainless-steel test coupons in the process water-flow lines in two paper machines operating in slightly alkaline conditions in temperatures (45 and 49 degrees C) supporting thermophilic microbes. Microbial succession was profiled using length heterogeneity analysis of PCR-amplified 16S rRNA genes (LH-PCR) and linking the sequence data of the created 16S rRNA gene libraries to the dominant LH-PCR peaks. Although the bacterial fingerprints obtained from the attached surface communities varied slightly in different samples, the biomarker signals of the dominating primary-colonizing bacterial groups remained high over time in each paper machine. Most of the 16S rRNA gene copies in the early biofilms were assigned to the genera Rhodobacter, Tepidimonas, and Cloacibacterium. The dominance of these sequence types decreased in the developing biofilms. Finally, as phylogenetically identical primary-colonizers were detected in the two different paper mills, the machines evidently had similar environmental conditions for bacterial growth and potentially a common source of contamination.

Deinococcus geothermalis has frequently been isolated from pink colored deposits of paper industry processes. Laboratory studies have shown that D. geothermalis is capable of forming on nonliving surfaces patchy biofilms that are resistant to adverse agents such as extreme pH, desiccation, solubilising detergents and biocides. This study was done to quantitatively assess the role of D. geothermalis as a biofouler in paper industry. Colored deposits were collected from 24 European and North American paper and board machines and the densities of the bacterial 16S rRNA genes and those of the red slime producers D. geothermalis and Meiothermus spp. were measured by QPCR (quantitative real time PCR). D. geothermalis was found at nine machines, usually from splash area deposits, but its contribution was minor, 0.001-1%, to the total bacterial burden of 8.3 to log 10.5 log units per gram wet-weight of the deposits. When D. geothermalis was found in a measurable quantity, Meiothermus spp. also was found, often in bulk quantity (7-100% of the total bacteria). The data are in line with the properties of D. geothermalis known from laboratory biofilm studies, indicating this species is a pioneer coloniser of machine surfaces and may help other bacteria to adhere and grown into biofilms, rather than competing with them.

The aim of this study was to determine the effect of varying nutrient conditions on biofilm formation of a Pseudomonas putida strain isolated from a paper machine under controlled conditions. Biofilm accumulation, was investigated using a laminar flow cell reactor in a defined mineral medium. Our results indicate that increasing nutrient concentration (from 0.1 to 0.5 gl(-1) glucose, C/N=40, C/P=100) or phosphate concentration (from C/P=200 to C/P=100) increased the rate and extent of biofilm accumulation, however, higher nutrient (1 gl(-1) glucose, C/N=40, C/P=100) or phosphate (C/P=50) concentration reduced biofilm accumulation rate because of a higher detachment. The rate and extent of biofilm accumulation increased with nitrogen concentration (from C/N=90 to C/N=20). Detachment is a key parameter that influences biofilm accumulation since the early stage (2h) of colonisation and strongly depends on nutrient conditions. In practice, controlling nutrient levels may be interesting to reduce biofilm formation in the paper industry.

To provide the first step towards a microbial ecology-based understanding of irreversible membrane biofouling, four laboratory-scale membrane bioreactors (MBRs) were operated to investigate the identity of bacterial populations highly correlated with irreversible membrane biofouling. The conventional MBR was divided into two separate experimental units. Unit one consisted of four suspended-growth, activated sludge, sequencing batch bioreactors treating a synthetic paper mill wastewater. Unit two consisted of a microfiltration membrane cell. Amplified ribosomal deoxyribonucleic acid restriction analysis (ARDRA) was used to compare the predominant bacterial populations in samples of mixed liquor and irreversibly bound to the membrane surface. The results of ARDRA showed a significant difference between the planktonic and sessile bacterial communities suggesting that irreversible biofouling of microfiltration membranes may be more highly correlated to specific bacterial populations rather than the total, bulk concentration of biomass. A custom-built mini-flow cell and light microscopy were used to visualise the early formation of biofilms by two pure cultures (Escherichia coli and Acinetobacter calcoaceticus) on membrane surfaces. The results confirmed that A. calcoaceticus was able to enhance the initiation of biofilm formation on microfiltration membranes.

This study reports on a multivariate analysis of the moving bed biofilm reactor (MBBR) wastewater treatment system at a Canadian pulp mill. The modelling approach involved a data overview by principal component analysis (PCA) followed by partial least squares (PLS) modelling with the objective of explaining and predicting changes in the BOD output of the reactor. Over two years of data with 87 process measurements were used to build the models. Variables were collected from the MBBR control scheme as well as upstream in the bleach plant and in digestion. To account for process dynamics, a variable lagging approach was used for variables with significant temporal correlations. It was found that wood type pulped at the mill was a significant variable governing reactor performance. Other important variables included flow parameters, faults in the temperature or pH control of the reactor, and some potential indirect indicators of biomass activity (residual nitrogen and pH out). The most predictive model was found to have an RMSEP value of 606 kgBOD/d, representing a 14.5% average error. This was a good fit, given the measurement error of the BOD test. Overall, the statistical approach was effective in describing and predicting MBBR treatment performance.

In this study, polysaccharide-producing bacteria were isolated from slimes collected from two Finnish and one Spanish paper mill and the exopolysaccharides (EPSs) produced by 18 isolates were characterised. Most of the isolates, selected on the bases of slimy colony morphology, were members of the family Enterobacteriaceae most frequently belonging to the genera Enterobacter and Klebsiella including Raoultella. All of the EPSs analysed showed the presence of charged groups in the form of uronic acid or pyruvate revealing the polyanionic nature of these polysaccharides. Further results of the carbohydrate analysis showed that the EPS produced by nine of the enterobacteria was colanic acid.

An investigation was conducted to explore the lignin-degrading capacity of attached-growth white-rot fungi. Five white-rot fungi, Phanerochaete chrysosporium, Pleurotus ostreatus, Lentinus edodes, Trametes versicolor and S22, grown on a porous plastic media, were individually used to treat black liquor from a pulp and paper mill. Over 71% of lignin and 48% of chemical oxygen demand (COD) were removed from the wastewater. Several factors, including pH, concentrations of carbon, nitrogen and trace elements in wastewater, all had significant effects on the degradation of lignin and the removal of COD. Three white-rot fungi, P. chrysosporium, P. ostreatus and S22, showed high capacity for lignin degradation at pH 9.0-11.0. The addition of 1 g l-1 glucose and 0.2 g l-1 ammonium tartrate was beneficial for the degradation of lignin by the white-rot fungi studied.

Thirty exopolysaccharides (EPS) produced by bacteria isolated from biofilms or slimelayers from different paper and board mills in Finland, France and Spain were subjected to size exclusion chromatography and sugar compositional analysis. High performance size exclusion chromatography (HPSEC) analysis revealed that some samples were composed of several molecular weight populations. These samples were fractionated by size exclusion chromatography and pooled accordingly. Principal components analysis (PCA) of the sugar compositions of the different pools indicated the presence of glucans and mannans caused by insufficient removal of the carbon or nitrogen source (yeast extract) from the bacteria growth medium leading to an overestimation of the glucose and mannose level in the sample, respectively. From the point of view of slime problems the EPS populations are the most important for multivariate analysis. Four groups of EPSs have been recognized by PCA analysis: a group of EPSs produced by Enterobacter and related genera similar to the regularly reported colanic acid; a group of Methylobacterium EPSs having high galactose and pyruvate levels and two groups that showed less dense clusters produced by Bacillus and related genera, showing high mannose and/or glucose levels and Klebsiella EPSs that showed galactose with rhamnose as major characteristic sugar moieties. Fourier transform infrared spectroscopy (FT-IR) of the same samples followed by discriminant partial least squares regression (DPLS) and linear discriminant analysis (LDA) showed that, when used with a well-defined training set, FT-IR could be used clustering instead of time-consuming sugar composition analysis. The Enterobacter and Methylobacetrium EPS groups could be recognized clearly. However the fact that this could hardly be done for the other two groups in the dataset indicates the importance of a larger and well-defined training or calibration set. The potential to use FT-IR, as a tool for pattern recognition and clustering with respect to EPS structures produced by micro organisms isolated from a paper mill environment is discussed.

Development of novel enzymatic methods for slime deposit control in paper mills requires knowledge of polysaccharide-producing organisms and the polysaccharide structures present in deposits. In this work, 27 polysaccharide-producing bacteria were isolated from slime samples collected from different parts of a paper machine. Most of the isolates produced polysaccharides in liquid culture and nine of them were selected for production of polysaccharides for characterisation. The selected isolates belonged to seven different genera: Bacillus, Brevundimonas, Cytophaga, Enterobacter, Klebsiella, Paenibacillus and Starkeya. Using ribotyping, partial 16S rDNA sequencing, physiological tests and fatty acid analysis, four of the nine isolates: Bacillus cereus, Brevundimonas vesicularis, K. pneumoniae and P. stellifer were identified to the species level. Production of polysaccharides by the selected isolates varied between 0.07 and 1.20 g L(-1), the highest amount being produced by B. vesicularis. The polysaccharides were heteropolysaccharides with varying proportions of galactose, glucose mannose, rhamnose fucose and uronic acids.

Parallel laboratory investigations were conducted to examine aspects of two distinct but related bioprocess strategies for low sludge production in the treatment of the same TCF kraft pulp mill effluent. The purpose of this article has been to compare the performance results from these two bench-scale trials with respect to nutrient demands, nutrient discharge, COD removal, and waste sludge characteristics. The LSP (Low Sludge Production) process can be used to significantly reduce sludge yield with excellent sludge characteristics. These sludge characteristics seemed to be related to elevated protozoan grazing pressures. The BAS (Biofilm-Activated Sludge) process achieves similar reduced sludge yields and sludge characteristics while at the same time significantly reducing the nutrient demands and discharge levels. For both LSP and BAS process optimization, the selector nutrient loading is critical to the overall process performance. Selector nutrient requirements are distinct from the overall process nutrient requirements.

Thermophilic aerobic treatment of settled pulp and paper mill effluent was studied under mill premises with two comparative pilot processes; suspended carrier biofilm process (SCBP) and activated sludge process (ASP). Full-scale mesophilic activated sludge process was a reference treatment. During the runs (61 days) hydraulic retention times (HRTs) were kept 13+/-5 h and 16+/-6 h for SCBP and ASP, respectively. Corresponding volumetric loadings rates (VLR) were 2.7+/-0.9 and 2.2+/-1.0 kg CODfilt m(-3)d(-1). Temperatures varied between 46 to 60 degrees C in both processes. Mesophilic ASP was operated with HRT of 36 h, corresponding VLR of 0.7 kg CODfilt m(-3)d(-1). Both SCBP and ASP achieved CODfilt (GF/A filtered) removals up to 85%, while the mesophilic ASP removal was 89+/-2%. NTU values were markedly higher (100-300) in thermophilic effluents than in mesophilic effluent (30). Effluent turbidity was highly dependent on temperature; in batch experiment mesophilic effluent sample had NTU values of 30 and 60 at 35 degrees C and 55 degrees C, respectively. As a conclusion, both thermophilic treatments gave high CODfilt removals, which were close to mesophilic process removal and were achieved with less than half of HRT.

A process of advanced oxidation treatment discussed here has been developed in the last seven years. A technical plant for treatment of paper mill wastewater started operation in 1999. Advanced oxidation treatment is defined here as a combination of chemical and biochemical oxidation applied to a completely biodegraded effluent. The process is a combination of ozonation with following biodegradation in a biofilm reactor. The combination makes use of the effect of partial oxidation in which with reduced use of expensive chemical oxidants persistent COD becomes biodegradable. A far-reaching elimination of AOX, colour and other disturbing substances like complexing agents is achieved simultaneously. The partially oxidized compounds, now biodegradable, are eliminated in a following biofilm reactor. Using single-stage systems, COD elimination rates of up to 60% are achieved with 0.4 to 1.0 g ozone per g COD eliminated in the combined process. With a two-stage system COD elimination of 85% with 0.65 g/g has been achieved in pilot tests. The quality of the treated effluent suits fresh water requirements for most types of paper production, so that either a total closure of the water circuits or - due to increasing concentrations of electrolytes, which are not eliminated by this process - a substantial closure to about 20% of the original amount of fresh water can be achieved.

A combination of the suspended carrier biofilm process and the activated sludge process (biofilm-activated sludge--BAS) has been shown to be very successful for the treatment of different types of pulp and paper mill effluents. The robust biofilm pre-treatment in combination with activated sludge results in a stable, compact and highly efficient process. Recent findings have shown that nutrient limited operation of the biofilm process greatly improves the sludge characteristics in the following activated sludge stage, while minimising sludge production and effluent discharge of nutrients. The nutrient limited BAS process was implemented at full scale at the Södra Cell Värö kraft mill and taken into operation in July 2002. After start-up and optimisation over about 5 months, the process meets all effluent discharge limits. The removal of COD is close to 70% and the removal of EDTA greater than 90%. Typical effluent concentrations of suspended solids and nutrients during stable operations have been 20-30 mg/L TSS, 0.3-0.5 mg/L phosphorus and 3-5 mg/L nitrogen. The sludge production was 0.09 kgSS/kg COD removed and the sludge volume index was 50-100 mL/g.

A two-stage pilot-scale thermophilic aerobic suspended carrier biofilm process (SCBP) was set up for the on-site treatment of pulp and paper mill whitewater lining. The microbial diversity in this process was analyzed by length heterogeneity analysis of PCR-amplified 16S ribosomal DNA. The primer pair selected for PCR amplification was first evaluated by a computational analysis of fragment lengths in ten main phylogenetical eubacterial groups. The fragment contained the first third of the 16S rRNA gene, which was shown to vary naturally between 465 and 563 bp in length. The length heterogeneity analysis of polymerase chain reaction (LH-PCR) profile of the biomass attached to carrier elements was found to be diverse in both stages of the SCBP. During normal operating conditions, sequences belonging to beta-Proteobacteria, Cytophaga/Flexibacter/Bacteroides group and gamma-Proteobacteria were assigned to the most prominent LH-PCR peak. Samples from the suspended biomass consisted of completely different bacterial populations, which were, however, similar in the serial reactors. The pilot process experienced alkaline shocks, after which Bacillus-like sequences were detected in both the biofilm and suspended biomass. However, when the conditions were reversed, the normal microbial population in the biofilm recovered rapidly without further biomass inoculations. This study shows that LH-PCR is a valuable method for profiling microbial diversity and dynamics in industrial wastewater processes.

Biofilms cause several problems in papermaking. This report describes a microbiological survey of colored biofilms in six paper and board machines, including two case studies of outbreaks of colored slimes in which the causative bacteria were found. A total of 95 pink-, red-, orange- or yellow-pigmented strains were isolated. Nearly all (99%) of the strains grew at 52 degrees C, 72% grew at 56 degrees C, but only 30% grew at 28 degrees C, indicating that most of the strains were moderately thermophilic. Biofilm formation potential and biocide susceptibility of the strains were analyzed with a microtiter plate assay. In the presence of 5 ppm of methylene bisthiocyanate or 2,2-dibromo-3-nitrilopropionamide in paper-machine water, 55 strains formed biofims. Moreover, 39 strains increased biofilm production by 5-753% in the presence of biocide, suggesting that biocide concentrations inhibitory to planktonic but not to surface-attached cells may actually promote biofouling. The cells may have inactivated a portion of the biocides, as the cell density in this assay was high, corresponding to the highest cell densities occurring in the circulating waters. Four groups of colored bacteria that were isolated from several mills were identified. Pink-pigmented Deinococcus geothermalis and red-pigmented Meiothermus silvanus occurred as common primary biofilm-formers in paper machines. This report is the first description of the involvement of Meiothermus species in red-slime formation in the paper industry. The third group of bacteria (putative new species related to Roseomonas) contained strains that were not biofilm formers, but which were commonly found in slimes of neutral or alkaline machines. The fourth group contained red-pigmented biofilm-forming strains representing a novel genus of alpha- Proteobacteria related to Rhodobacter. Many colored paper-machine bacteria are species previously known from microbial mats of hot springs. Some characteristics of the bacterial groups are described here in order to facilitate their recognition in future cases of colored-slime outbreaks in the paper industry.

Paper machine biofilms formed in situ on stainless steel surfaces were studied. A robust flow cell was fitted to side stream (1.8 m s(-1)) of the spray water circuit of a paper machine. This on-site tool allowed for assessing the efficacy of antifoulants and the adequacy of steel polishing under mill conditions. A rapid fluorescence-based assay was developed to quantify the biomass of shallow biofilms on machine steel. The fluorescence matched the ATP content measured for the same biofilms. Electrolytic polishing reduced the tendency of biofouling of 500 grit surface steel. Biofilm grew under machine conditions as clusters on the steels, showing uniformly coccoid, filaments or short rods; only one cell type in each cluster. The biofilm clusters excluded latex beads of 0.02 microm with hydrophilic or with hydrophobic surfaces from penetrating more than three to four layers of cells. Under the high hydraulic flow at the machine (1.8 m s(-1)), the biofilm grew in 7 days 6-10 microm thick. The high flow rate guided the shape of the biofilm clusters emerging after the primary attachment of cells. Adhered individual bacteria were the platform on steel to which solids such as paper machine fines then accumulated.

Mechanisms for the undesired persistence of Bacillus species in paper machine slimes were investigated. Biofilm formation was measured for industrial Bacillus isolates under paper machine wet-end-simulating conditions (white water, pH 7, agitated at 45 degrees C for 1-2 days). None of the 40 tested strains of seven Bacillus species formed biofilm on polished stainless steel or on polystyrene surfaces as a monoculture. Under the same conditions, Deinococcus geothermalis E50051 covered all test surfaces as a patchy thick biofilm. The paper machine bacilli, however, formed mixed biofilms with D. geothermalis E50051 as revealed by confocal microscopy. Biofilm interactions between the bacilli and the deinococci varied from synergism to antagonism. Synergism in biofilm formation of D. geothermalis E50051 was strongest with Bacillus coagulans D50192, and with the type strains of B. coagulans, B. amyloliquefaciens or B. pumilus. Two B. licheniformis, one B. amyloliquefaciens, one B. pumilus and four B. cereus strains antagonized biofilm production by D. geothermalis. B. licheniformis D50141 and the type strain of B. licheniformis were the strongest antagonists. These bacteria inhibited deinococcal growth by emitting heat-stable, methanol-soluble metabolite(s). We conclude that the persistence of Bacillus species in paper machine slimes relates to their ability to conquer biofilms formed by primary colonizers, such as D. geothermalis.

Biofilms of paper mill bacteria were cultivated in paper mill white water-simulating conditions on glass slides or stainless steel coupons in a laboratory culture system. The sugar content and composition of the biofilms were analysed and compared with the sugar composition of paper mill slimes. Acid methanolysis followed by gas chromatography revealed that Burkholderia was the major biofilm producer in pure culture, producing up to 50 microg of biofilm sugar cm(-2) in 5 days in rich medium and 10 microg in paper mill simulating medium. A mixture of simulated paper mill water with a culture medium yielded more biofilm (100 microg cm(-2)) than either of the media alone, so the biofilm accumulation was not proportional to the available substrate. More biofilm accumulated on stainless steel coupons than on glass slides, and the steel-coupon biofilms contained slightly more uronic acids. The biofilm sugars contained mainly galactose, glucose, mannose, and rhamnose. In paper mill medium, the Burkholderia biofilm contained more galactose and glucose, and less rhamnose, than in rich laboratory medium. The sugar composition of paper mill slimes was quite similar to those of steel-cultured Burkholderia cepacia biofilms. This suggests that Burkholderia cepacia is responsible for much of the slime in the paper mill.