Diversity of bacteria contaminating paper machines

Formation of microbial biofilms and slimes is a general and serious problem in the operation of paper machines. Studies of microbial populations in paper machine-derived biofilms have been conducted using standard microbiological procedures; however, the bacterial genera present in this type of samples as well as their diversity are quite poorly known. Here, the bacterial diversity of 38 process water and 22 biofilm samples from four different Finnish paper machines were analyzed by length heterogeneity analysis of PCR-amplified 16S ribosomal DNA (LH-PCR). In addition, sequencing of the amplified 16S rRNA gene from 69 clones was conducted for characterization of the bacterial genera present in biofilm and slime samples. The LH-PCR profiles of both the free-living (process waters) and immobilized (biofilms) bacteria were diverse at all stages of the papermaking process. Out of the 69 sequenced clones, 44 belonged to alpha-Proteobacteria, most of which were close to the nitrogen-fixing root nodule genera Sinorhizobium, Rhizobium and Azorhizobium. Other clones were assigned to beta- and gamma-Proteobacteria and the phylum Bacteroidetes. In addition, eight of the clones were assigned to a yet uncultivated phylum, TM7. Finally, epifluorescence microscopy revealed that Gram-negative bacteria were predominant in both the biofilm (65%) and process water (54%) samples and a small coccoid cell morphology was most common in all samples. Together, our results show that the analysis of microbial samples from paper machines using modern molecular biology techniques adds valuable information and should, therefore, be useful as a more specific and sensitive microbiological method for the paper industry. This information could further be applied, e.g., in the development of more specific and environmental friendly antimicrobial agents for paper mills.     

Structure and on-site formation of biofilms in paper machine water flow

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 μm 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 μm 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. Journal of Industrial Microbiology & Biotechnology (2002) 28, 268–279 DOI: 10.1038/sj/jim/7000242 Received 04 October 2001/ Accepted in revised form 14 January 2002     

Electrochemical and microbiological characterization of paper mill biofilms

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.     

Mini-review: microbial problems in paper production

Paper mills are open systems, which provide favorable conditions for microbial growth. Microbial contamination can cause substantial economic losses, including the deterioration of raw materials, interference with production processes by breakdowns and lowering product quality, and eventually, problems in wastewater treatment. Damage is caused by acidification, attack on raw materials, the formation of odorous products, discoloration of pigments, and the formation of methane and hydrogen, thereby producing potentially explosive conditions. Population analyses have revealed that a wide variety of microorganisms are involved, but there appear to be no typical strains associated with paper mills. Current trends in process engineering, such as chlorine-free bleaching, processing at neutral pH, closed cycles, and the use of recycled paper also favor microbial growth and biofilm (slime) formation. A fundamental problem associated with slimes is the extensive matrix of extracellular polymeric substances, which is composed of a large variety of highly hydrated polysaccharides, proteins, nucleic acids, and lipids. No ‘silver bullet’ against biofouling can be expected, and effective countermeasures have to be based on holistic approaches.     

Molecular characterization of the bacterial communities in the different compartments of a full-scale reverse-osmosis water purification plant

The origin, structure, and composition of biofilms in various compartments of an industrial full-scale reverse-osmosis (RO) membrane water purification plant were analyzed by molecular biological methods. Samples were taken when the RO installation suffered from a substantial pressure drop and decreased production. The bacterial community of the RO membrane biofilm was clearly different from the bacterial community present at other locations in the RO plant, indicating the development of a specialized bacterial community on the RO membranes. The typical freshwater phylotypes in the RO membrane biofilm (i.e., Proteobacteria, Cytophaga-Flexibacter-Bacteroides group, and Firmicutes) were also present in the water sample fed to the plant, suggesting a feed water origin. However, the relative abundances of the different species in the mature biofilm were different from those in the feed water, indicating that the biofilm was actively formed on the RO membrane sheets and was not the result of a concentration of bacteria present in the feed water. The majority of the microorganisms (59% of the total number of clones) in the biofilm were related to the class Proteobacteria, with a dominance of Sphingomonas spp. (27% of all clones). Members of the genus Sphingomonas seem to be responsible for the biofouling of the membranes in the RO installation.     

Plant-derived compounds as natural antimicrobials to control paper mill biofilms

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.     

Biofouling of Groundwater Systems by Thiothrix spp.

Thiothrix spp., sulfide-oxidizing filamentous bacteria, were found to be a principal bacterial component of aquatic biofilms causing biofouling in selected municipal water storage tanks, private wells, and drip irrigation systems in Florida. Treatments of up to 200 ppm chlorine in the affected systems could not prevent return of the biofouling problem. The water originated from the upper Floridan aquifer and associated surficial aquifers in central and north Florida. Samples were examined where visible biofilms had a white, filamentous appearance, indicative of Thiothrix spp. The detection of Thiothrix spp. was confirmed by enzyme-liked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and microbiological procedures. It was estimated through immunocytochemical procedures that Thiothrix spp. comprised 18% of the biofilm in the municipal water storage tanks. These observations confirm that specific biological and chemical interactions may induce physical changes leading to significant biofouling. Received: 6 November 1996 / Accepted: 14 March 1997     

Detection and quantitation of colored deposit-forming <Emphasis Type="Italic">Meiothermus</Emphasis> spp. in paper industry processes and end products

Colored biofilms cause problems in paper industry. In this work we used real-time PCR to detect and to quantitate members of the genus Meiothermus from the process samples and end products from 24 machines manufacturing pulp, paper and board in four countries. The results obtained from 200 samples showed the importance of members of the genus Meiothermus as ubiquitous biofoulers in paper machines. This genus was the dominant biofouler in some mills. From ≤10⁴ to 10¹¹ copies of Meiothermus 16S rRNA genes were found per gram of process deposit (wet weight). Meiothermus spp. were found in paper and board products with colored defects and connection between deposit-forming microbes and end-product spots was shown. 16S rRNA gene sequences of 29 biofilm producing bacterial isolates from different mills were determined. Based on sequence data, 25 of the isolates were assigned to the genus Meiothermus, with Meiothermus silvanus and M. ruber as the most frequent species.     

Prokaryotic microbiota of recycled paper mills with low or zero effluent

The tendency in the paper industry is to close all water loops to save water. This leads to higher process temperatures and may increase the colloidal and dissolved material in the process circulation. Increase of nutrients in the water circuits may favor microbiological growth and fouling. In this paper the chemical and microbial compositions of water circuits and deposits were studied of two closed cycled paper/board mills, one mill totally closed (0 m(3) waste water t(-1)), and the other low discharging (about 4 m(3 )t(-1)). The zero discharge mill accumulated high amounts (>10 g C L(-1)) of organic carbon in the circulation waters, about 40% of which composed of volatile acids (lactic, acetic, propionic and butyric acid). Water contents of sulfate, chloride, sodium and calcium increased to >1 g L(-1) of each. q-PCR targeted on 16S rRNA genes indicated that the bacteria in water circuits were mainly viable cells. In both mills anaerobic growth (10(6)-10(8) CFU mL(-1)) equalled or exceeded aerobic growth, with odor problem but no actual slime problem. The major part (40%) of all identifiable bacterial sequences were closest but yet distant (<96%) to Enterococcus cecorum and in the 4 m(3 )t(-1) discharging mill also Bacillus thermoamylovorans and Bacillus coagulans. Slimes and deposits from the mills contained high amounts, >/=10(8) g(-1), of archaean, but only the genus Methanothrix was identifiable from the cloned sequences. The findings indicate that closing the water circuits strongly limited diversity of the microbiota but allowed efficient mineralization of the dissolved and suspended matter.     

Characterisation of aerobically grown non-spore-forming bacteria from paper mill pulps containing recycled fibres

A total of 179 non-spore-forming bacteria aerobically growing on Nutrient Agar, Plate Count Agar or in specific enrichment conditions for salmonella, campylobacteria, listeria, yersinia or staphylococci, were isolated from 16 untreated paper mill pulps. After phenotypical screening the isolates were characterised by automated ribotyping and partial sequencing of the 16S rRNA gene. They could be divided into seven taxonomical classes representing 63 taxa (species): actinobacteria (11 species), bacilli (7), flavobacteria (3) alphaproteobacteria (10), betaproteobacteria (5), gammaproteobacteria (25) and sphingobacteria (2). Most of the gammaproteobacteria were enterobacteria, mainly species of the genera Enterobacter (7 species, 7 samples/3 mills) and Klebsiella (5 species, 6 samples/3 mills). Other commonly occurring bacteria were most closely related to Microbacterium barkeri (7 samples/3 mills), Cloacibacterium normanense (6 samples/2 mills), Pseudoxanthomonas taiwanensis (5 samples/2 mills) and Sphingobacterium composti (5 samples/1 mill). Sporadic isolates of Listeria innocua, L. monocytogenes, Enterococcus casseliflavus and Staphylococcus warneri were detected, from which only L. monocytogenes is considered to be a food pathogen. No isolates of the genera Campylobacter, Salmonella or Yersinia were detected. The detected bacteria may be harmful in process control, but the load of food pathogens with recycled fibres to paper machines is insignificant. Faecal contamination of the pulp samples was not indicated.     

Heterogeneity and spatial distribution of bacterial background contamination in pulp and process water of a paper mill

Identifying the source and the distribution of bacterial contaminant communities in water circuits of industrial applications is critical even when the process may not show signs of acute biofouling. The endemic contamination of facilities can cause adverse effects on process runability but may be masked by the observed daily variability. The distribution of background communities of bacterial contaminants may therefore be critical in the development of new site-specific antifouling strategies. In a paper mill as one example for a full-scale production process, bacterial contaminants in process water and pulp suspensions were mapped using molecular fingerprints at representative locations throughout the plant. These ecological data were analyzed in the process–engineering context of pulp and water flow in the facilities. Dispersal limits within the plant environment led to the presence of distinct groups of contaminant communities in the primary units of the plant, despite high flows of water and paper pulp between units. In the paper machine circuit, community profiles were more homogeneous than in the other primary units. The variability between sampled communities in each primary unit was used to identify a possible point source of microbial contamination, in this case a storage silo for reused pulp. Part of the contamination problem in the paper mill is likely related to indirect effects of microbial activity under the local conditions in the silo rather than to the direct presence of accumulated microbial biomass.     

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     

Sugar composition of biofilms produced by paper mill bacteria

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.     

Biofilm diatom community structure: Influence of temporal and substratum variability

Abstract

Diatoms, which are early autotrophic colonisers, are an important constituent of the biofouling community in the marine environment. The effects of substratum and temporal variations on the fouling diatom community structure in a monsoon-influenced tropical estuary were studied. Fibreglass and glass coupons were exposed every month for a period of 4 days and the diatom population sampled at 24 h intervals, over a period of 14 months. The planktonic diatom community structure differed from the biofilm community. Pennate diatoms dominated the biofilms whilst centric diatoms were dominant in the water column. Among the biofilm diatoms, species belonging to the genera Navicula, Amphora, Nitzschia, Pleurosigma and Thalassionema were dominant. On certain occasions, the influence of planktonic blooms was also seen on the biofilm community. A comparative study of biofilms formed on the two substrata revealed significant differences in density and diversity. However species composition was almost constant. In addition to substratum variations, the biofilm diatom community structure also showed significant seasonal variations, which were attributed to physico-chemical and biological changes in both the water and substratum. Temporal variations in the tychopelagic diatoms of the water were also observed to exert an influence on the biofilm diatom community. Variations in diatom communities may determine the functional ecosystem of the benthic environment.     

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.     

Factors regulating the density of bacteria in process waters of a paper mill

Variations in the numbers of total colonies of Klebsiella pneumoniae, Acinetobacter spp. and pseudomonads were investigated in process waters of a paper mill in southern Finland. Variations were related to independent parameters, namely temperature, pH, redox potential and production of offset paper, by using multiple regression analysis. Temperature was the most significant regressor variable and was negatively correlated with bacterial counts. It accounted for up to about 80% of the variance in bacterial counts in various parts of the process. The significance of temperature was due to its fluctuations in a critical range, above and below the maxima for bacterial growth. The pH level was also significant for total colony count and for K. pneumoniae. Redox potential and the production of offset paper were of significance for Acinetobacter spp. Washing the paper machine with water and lye decreased the numbers of bacteria in process waters.     

Dominance of endospore-forming bacteria on a Rotating Activated Bacillus Contactor biofilm for advanced wastewater treatment

The bacterial diversity inherent to the biofilm community structure of a modified rotating biological contactor wastewater treatment process, referred to as the Rotating Activated Bacillus Contactor (RABC) process, was characterized in this study, via both culture-dependent and culture-independent methods. On the basis of culture-dependent methods, Bacillus sp. were found to exist in large numbers on the biofilm (6.5% of the heterotrophic bacteria) and the microbial composition of the biofilms was quite simple. Only three phyla were identified-namely, the Proteobacteria, the Actinobacteria (High G+C Gram-positive bacteria), and the Firmicutes (Low G+C Gram-positive bacteria). The culture-independent partial 16S rDNA sequence analysis revealed a considerably more diverse microbial composition within the biofilms. A total of eight phyla were recovered in this case, three of which were major groups: the Firmicutes (43.9%), the Proteobacteria (28.6%), and the Bacteroidetes (17.6%). The remaining five phyla were minor groups: the Planctomycetes (4.4%), the Chlorobi (2.2%), the Actinobacteria (1.1%), the Nitrospirae (1.1%), and the Verrucomicrobia (1.1%). The two most abundant genera detected were the endospore-forming bacteria (31.8%), Clostridium and Bacillus, both of which are members of the Firmicutes phylum. This finding indicates that these endospore-forming bacteria successfully colonized and dominated the RABC process biofilms. Many of the colonies or clones recovered from the biofilms evidenced significantly high homology in the 16S rDNA sequences of bacteria stored in databases associated with advanced wastewater treatment capabilities, including nitrification and denitrification, phosphorus accumulation, the removal of volatile odors, and the removal of chlorohydrocarbons or heavy metals. The microbial community structures observed in the biofilms were found to correlate nicely with the enhanced performance of advanced wastewater treatment protocols.     

A survey of the composition and diversity of bacterial populations in bleached kraft pulp-mill wastewater secondary treatment systems

Bacterial community compositions from 10 pulp- and paper-mill treatment systems were compared using both traditional and molecular techniques. 16S-RFLP (Random Fragment Length Polymorphisms) analysis was used to examine the genotypic profiles of the whole bacterial community of each treatment system. Although all the communities shared approximately 60% of their DNA band pattern, as determined by computer-assisted cluster analysis, each community displayed a unique profile that was stable over time under normal operating parameters. Reverse Sample Genome Probing (RSGP) and 16S-RFLP were used to compare the culturable bacterial communities of several geographically separated pulp-mill biotreatment system communities. There was little overlap in the composition of the culturable community between mills at the genus level. Furthermore, RSGP variation was almost as high within a mill as between mills. Partial sequences of the 16S rRNA genes from culturable isolates identified Bacillus spp., Pseudomonas spp., and Xanthobacter as some of the dominant species. Finally, several 16S rRNA genes from two whole community 16S RNA gene libraries were partially sequenced and identified as similar to unknown alpha-, beta-, and gamma-Proteobacteria, Ralstonia, Alcaligenes, Nitrospira, Firmicutes, and clones representing the new Holophaga/Acidobacterium phylum. These findings suggest that although these pulp- and paper-mill biotreatment communities perform similar functions, they are populated by unique mixtures of species.     

Microbial incidence in upper respiratory tracts of workers in the paper industry.

The incidence of microbes in the nasal cavities of workers in three paper and board mills was investigated. A total of 234 persons exposed to microbial aerosols and splashes from paper machine wires and debarker drums formed the exposed group. The control group consisted of 294 workers from the dry working areas: the winding and packing sections. Chi-square analysis was used to test the differences in the frequency of microbial incidence and various symptoms between the exposed and control groups. The nasal cavities of many workers, particularly workers in the debarkers, proved to be contaminated by Klebsiella pneumoniae, other coliforms, yeasts, and molds; usually only one microbe was involved, but sometimes two or several species were found. Nasal bacteria and yeasts were largely derived from the mill and debarker air; the microbes in the air came mainly from process waters. Lack of association of nasopharyngeal symptoms with either exposure to aerosols or nasal microbial contamination was interpreted as an indication of host defenses that were adequate to protect workers from harmful microbial colonization in paper mill environments.     

Application of enzymes in the pulp and paper industry

The pulp and paper industry processes huge quantities of lignocellulosic biomass every year. The technology for pulp manufacture is highly diverse, and numerous opportunities exist for the application of microbial enzymes. Historically, enzymes have found some uses in the paper industry, but these have been mainly confined to areas such as modifications of raw starch. However, a wide range of applications in the pulp and paper industry have now been identified. The use of enzymes in the pulp and paper industry has grown rapidly since the mid 1980s. While many applications of enzymes in the pulp and paper industry are still in the research and development stage, several applications have found their way into the mills in an unprecedented short period of time. Currently the most important application of enzymes is in the prebleaching of kraft pulp. Xylanase enzymes have been found to be most effective for that purpose. Xylanase prebleaching technology is now in use at several mills worldwide. This technology has been successfully transferred to full industrial scale in just a few years. The enzymatic pitch control method using lipase was put into practice in a large-scale paper-making process as a routine operation in the early 1990s and was the first case in the world in which an enzyme was successfully applied in the actual paper-making process. Improvement of pulp drainage with enzymes is practiced routinely at mill scale. Enzymatic deinking has also been successfully applied during mill trials and can be expected to expand in application as increasing amounts of newsprint must be deinked and recycled. The University of Georgia has recently opened a pilot plant for deinking of recycled paper. Pulp bleaching with a laccase mediator system has reached pilot plant stage and is expected to be commercialized soon. Enzymatic debarking, enzymatic beating, and reduction of vessel picking with enzymes are still in the R&D stage but hold great promise for reducing energy. Other enzymatic applications, i.e., removal of shives and slime, retting of flax fibers, and selective removal of xylan, are also expected to have a profound impact on the future technology of the pulp and paper-making process.