Trends in environmental microbiology with references from paper industry microbiology

The history of microbiology contains several eras with different targets. I will refer them in this way (based on my over 30 years experience as microbiologist and teacher of microbiology and biochemistry):

In the beginning, cultivation and observation of microorganisms was the main target. Doctors like Pasteur and Koch were very innovative and developed intelligent culture medias and vessels to perform very delicate experiments. The everlasting fight against pathogenic microbes was the primary target but Louis Pasteur started to help eg. wine producers to solve their quality problems, caused by microbes.

The combination of microbiology and biochemistry on the second era was very satisfying by solving questions concerning the huge amount of anabolic and catabolic processes included in microbial growth. More and more were also learned in the area of microbial ecology. Questions like "who? what? when? where? how? why?" were partially solved (ref. MADSEN,E.L. 2008. Environmental Microbiology. From Genomes to biochemistry. Blackwell Publishing).

"Third era" can be described by the novel methods to identify bacteria. Biochemical test kits (API etc.) were replaced by Fatty Acide Methylated Ester method (FAME) by Hewlett-Packard on 80's. After it, molecular biology methods, based on ribosomal RNA and DNA, helped to construct the development lines of microorganisms.

Today is the time of new era. We know the "family trees" of bacteria but we should now continue with environmental microbiology and microbial ecology to solve questions like "How, why, by whom and in which conditions will the raw materials of paper industry be biodeteriorated?", "How can we prevent these processes by setting the process conditions unsuitable for those biochemical processes?", "Can we prevent the growth of biofilms and slimes in an ecological way?", "How to prevent selectively the growth of toxin producers like Bacillus cereus in paper and board processes?", how to fight against Legionella in paper industry?".

Names are not the most important thing. Most important is, how the bacteria act in different ecological niches of a paper machine. This work has to be done by using simulations of paper processes which is possible by wet end simulators of research units (as an example: VTT in Jyväskylä, Finland) and laboratory/field instruments (like biofilm detectors in the processes or PMEU incubators by Samplion Ltd.).

The role of PMEU is getting more and more important because this method helps to detect microbial growth of different types (biofilms included) in a very short period of time as well as to test simultaneously the effects of alternative biocides in small-scale tests whose growth parameters match with the growth conditions in the real processes.

We are - and we shall - turn back to the era of Pasteur & Koch: the names are already known, and we shall now investigate, what the contaminating microbes are doing in the industrial processes and how to prevent losses of raw material, machine stops and poor quality of the products by simulating growth processes in small-scale tests, performed in the laboratory or in the field, by the machies themselves.

Connections of paper industry microbiology to other sectors of microbiology: what is actually needed?

To make any definitions of paper industry microbiology, it makes sense to compare it with elder sectors of microbiology. Despite the microbiological problems of the paper processes and the paper products have been obvious since the beginning of machine-scale production of paper and board on 19th century, their effects have get worser when the scale, speed and raw material repertoir have increased during last decades. The tradition of the microbiological control, as well as the history of biocide research, intended in the "healthcare" of paper and board machines is therefore much shorter than in related areas like in dairy or food microbiology.

Paper manufacturing processes could be seen as ecosystems where several, complicated microbiological processes are continuing day and night. Microbial communities perform their important role as the actors of chemical transformations which shall modify most living and very many non-living substances into forms which will support the growth of other living creatures. Many species of immigrant bacteria, coming into the processes with the raw water, mechanical fibres and several additives, will feel fine: favourable temperature, pH level and nutrient concentrations, as well as good aeration and a huge supply of contact surfaces to build up biofilms, are available for them. They really do not make any difference between their lives outside and inside of the paper mill walls.

Unfortunately (not for the microbes but for the paper production) there are some features of paper machines which are similar with fermentor and bioreactor processes of biotechnological industry. So many growth factors (some of them were mentioned above) will be kept on so controlled levels that the adaptation of certain microbiological population cannot be avoided. It shall also be kept in mind that the long running periods will increase the microbiological risks by allowing long growing periods of microbes inside the machines.

How to control these problems?

Measures to dose biocidic compounds into the processes cannot be avoided because the conditions of paper and board machines cannot be adjusted on biocidic levels: the rise of the overall temperature over +80 oC is impossible, like the rise of pH value over 12. Before significant technical improvements to prevent the microbial growth in the paper machine processes could be done (if ever), the biocidic treatments and their rapid control methods like ON LINE biofilm measurements and frequent (at least once per 8 hours) AT LINE microbiological control of the main contaminating routes, wet end processes and towers containing white waters, pulps and brokes are the most important tools to secure the runnability of the machines and the quality of the products.

With the price of only 2-3 jumbo rolls can reliable instruments for the AT LINE microbiological control of the wet end processes be bought today. Alternative methods, many of them representing molecular biology methods, are available, but those which can show not only the counts of certain species but also the overall metabolic activities of the waterborne microbes and their potential to produce biofilms should be preferred. A combination of PMEU incubations and ATP Assays, with the addition of PCR if needed, is the most recommended procedure to show the effects of biocides on the microbial activity. PMEU method can be applied to biofilm testing, too.

The most important thing is that not only the counts of microbes (how high they may ever been) but also their overall metabolic activity and certain actions like breakdown of starches by amylase enzymes or production of H2S and H2 in anaerobic conditions shall be controlled all the time when the machines are running. All the laws of microbial ecology are present both in the nature and inside the machines - and they can lead to severe problems if counteracting does not work.

Transfer of microbiological control from institutes to mill labs

Rapid development of analytical microbiology has been obvious during last 20 years.

After the beginning of IM's career in paper industry (est. 1982) a significant increase of novel methods has taken place. Slow and labourous colony count analyses have been replaced with novel, advanced methods in certain laboratories on 1990's.

Biomass, surface hygiene, condition of activated sludge, biofilm formation - among even more subjects - can be assayed by luminometric methods today.

Light and UV microscopy is another basic tool of paper industry microbiology today. Very valuable results have been achieved with TEM on 1980's and articles about sporeforming bacteria and biofilms, based on electron microscopy, have published by researcher all over the world. TEM is, however, such an advanced research instrument which is practically impossible to apply into everyday microbiological control of pulp and paper mills. Confocal microscopy has given brand new ideas about the structure of biofilms but it is also a too complicated method for mill labs. In opposite, light and epifluorescence microscopy aren't too expensive; they definitely need a lot of training for the personnel which is no big problem, however: in Finland (and IM is sure, in other countries, too) are training companies who will have annual microbiology courses for paper industry under titles like "Paper Industry Microscopy" and "Methods for Process and Product Hygiene in Paper Industry".

Tools of molecular biology have replaced the previous generation's major tool, FAME (Fatty Acid Methylated Esters - an application of gas chromatography to perform identifications of bacteria). But the limits of PCR and similar methods are obvious: they cannot show what is really happening inside the machines! They only give - valuable, of course - information about microbial species but do not explain and forecast those metabolic reactions, succession of population, risk of biofilm formation etc. which are more important for the drive of machines, good housekeeping of raw materials and product hygiene.

Basic methods of modern microbiology like DEFT, ATP Assay, PCR and other should therefore be combined with simple simulators, driven in mill labs. This is already possible: the first system for this target, PMEU (Portable Microbiological Enrichment Unit) has been tested and used by IM since the beginning of 2000's and it has been proven to be a most valuable tool for rapid raw material, process and biofilm studies today.

An ecological point of view shall be applied to everyday mb control of the pulp and paper mills. This question is discussed in the article "Paperikone - ekosysteemi ja bioreactori" ("Paper Machine - An Ecosystem and A Bioreactor") by JM in the annual of Finnish Microbiology Society (INOCULA 2007 / 1 - unfortunately only in Finnish). Understanding of these two natures of a paper machine gives new chances for the mills: it gives the ability to forecast microbiological events inside the processes and it also give extra time to prevent problems.