Characterisation of Planktonic Microbial Populations in Paper-Mill Water Systems
Report No: 1459/1/07
December 2007


Due to the shortage of water, paper mills in South Africa are under pressure to use less water than their counterparts in Europe and North America.  Integrated water management plans for paper mills include strategies to reduce water consumption by closure of water circuits to reuse water.  Closure, however, directly and indirectly results in an increase in populations of microorganisms.  

The current practice for the monitoring of microbial populations, is for the industry to enumerate one or two guilds of microorganisms at a limited number of points.  Specific organisms are only identified in exceptional circumstances.  Comprehensive characterisation and identification of microbial populations could result in improved control and will extend the limits for mill closure.  Microbiological data could also aid in the prevention of biofilm formation and minimise corrosion and furthermore be useful to minimise health risks and improve efficiency of water treatment processes.  

The microbiologically associated problems that frequently occur in paper mills depend primarily on the degree of closure of the water system. Upon closure of the water system, the temperatures are frequently elevated and the nutrient concentrations generally increase.  As a result of the increased recycling of process water, nutrient salts and degradable carbon content increase, further contributing to the microbiologically associated problems such as biofilm formation and microbial induced corrosion. Microbiological control is, therefore, an important factor in reducing water consumption, health risks and maintenance costs as well as increasing production.  

The identification of microorganisms and characterisation of microbial populations is important for the design of microbial control programmes. Knowledge of the different microorganisms present in water systems and the interactions in populations would assist in the selection and dosage of the correct biocides to minimise microbial build-up.  Identification of microorganisms is also important to manage system upsets such as acidification of stock tanks and in recognition of health hazards. A microbial database that includes characteristics of populations and environmental conditions of their habitat could improve management of these water systems and result in the reduction of water consumption, production costs and health risks and improve effluent quality through efficient microbial control.  

The objectives of this project were, therefore, to compile a database of microorganisms and population characteristics in relation to physical properties of water systems in order to underpin effective water management in paper mills.  A further aim was to establish a facility for routine identification of microorganisms from industrial water systems and finally to train technical staff and students to provide a microbiological service to the pulp and paper industry.

Literature survey
A literature review was carried out with the objective to describe the environment in paper-mill water systems and to evaluate the influence of different physical parameters on microbial populations. The influence of microorganisms, especially corrosive biofilms, was discussed as well as different methods to minimise their effect in the water system of a typical paper mill.  Traditional and emerging techniques to quantify microorganisms in environmental samples were evaluated as well as the application of these techniques to assess biocide programmes.  It was concluded that control measures are necessary to counter the colonisation of microorganisms in paper mills, especially when water systems in paper machines are being closed to reduce water consumption. By monitoring different environmental parameters, microbial problems can be identified.  It is necessary to employ suitable counting procedures to enumerate microorganisms in any water system and plate counts remain the most popular method in industry due to low cost and ease of application. Plate counts are, however, time-consuming and biased. The following of trends of plate counts can indicate whether there is a difference in biological activity at different sampling points.  Using adenosine triphosphate (ATP) to indicate biological activity requires specialised equipment and expertise, but can be effective and time-saving when applied properly.

Over the past three years, environmental parameters were measured and samples collected during two surveys at each of the following paper machines: Two machines at Adamas (Port Elizabeth), two at Ngodwana (close to Nelspruit), three at Enstra (Springs), two at Stanger, one at Cape Kraft (Cape Town), four at Tugela (Mandini) and one paper machine at Saiccor (Umkomaas). These surveys produced data on 13 environmental variables, three microbial guilds and two diversity indices.  The interaction between environmental factors was found to be too complex to determine its effect on microbial populations and the data were subjected to multiple correlation analyses.  

These analyses showed that the build-up of temperatures associated with mill closure could lead to increased microbial numbers and microbial control must be considered when planning modifications to mills. The expected increase in chemical oxygen demand, nitrogen and phosphorous as result of recirculation could further increase microbial loads. The inverse relationship, observed between microbial numbers and oxidation-reduction potential, provides a technique for rapid assessment of microbial levels and, since oxidation-reduction potential is also influenced by oxidizing biocides, it could also indicate the levels of active biocide.  Most of the environmental aspects had an influence on the microbial growth and diversity parameters and, since so many were involved, the highest single-factor correlations were between 0.3 and 0.4. More of the variation in microbial numbers could possibly be explained by including more than one factor with weighting in models.

Enterobacterial Repetitive Intergenic Consensus – Polymerase Chain Reaction (ERIC-PCR) was used as a typing technique to distinguish between the prevalent isolates from paper machines and sequencing of the 16S rDNA gene made it possible to identify many of the isolates. The most prevalent bacteria were identified as members of the genus Acinetobacter and these strains were also the most widely distributed. The prevalent isolates from all surveys were characterised using BiologŪ substrate-utilisation profiles and Restriction Fragment Length Polymorphisms (RFLPs). The data were entered in a computer database and key programme that can be widely distributed at relatively little expense. It is easy to install and can be operated intuitively. The database is unique in terms of its focus on bacteria from paper-mill environments and currently contains a limited number of strains, but these strains represent bacteria forming more than 90% of the culturable populations in the surveyed environments. Unknown isolates can be identified by entering either Biolog or RFLP data and a data match returns, morphological and cultural characteristics as well as information on the environment where the reference isolate was collected. The database is set up in such a way that users will be able to easily add information from their own studies.  

  1. A database of prevalent microorganisms supported by a key, using either a molecular fingerprint or physiological profile, has been developed. The database includes information on the habitat characteristics, distribution and possible impact of each strain.  The significance of changes in different environmental parameters on microbial numbers has been determined through multivariate analysis and the results could be used for more effective water management in paper mills. A number of reports and papers have been generated to facilitate technology transfer (Appendix A).
  2. A facility for routine identification of microorganisms from industrial water systems was established and used throughout the project, but maintenance of this laboratory cannot continue due to limited resources. Reference cultures and the database software have been supplied to Buckman Laboratories and the Department of Food Science (University of Stellenbosch) where it will be applied to further this objective. It was also recommended that the Biolog system be donated to Buckman Laboratories.
  3. A number of staff members and students have been trained during the course of the project (Appendix B) and could provide a microbiological service to the pulp and paper industry.  In case they do not find employment in this industry, they would be valuable to most other industries where water quality is important.
It is recommended that the database and key software be distributed as widely as possible and not only within the paper industry, but also to other industries where bacterial control and identification play a role. Environmental parameters in water systems influence microbial numbers and parameters such as temperature and oxidation-reduction potential should be used to predict microbial levels. These data will be invaluable for integrated water management and especially when water systems are to be closed.