Future methods for P&P microbiology.

It seems that new methods for P&P microbiology are needed.

After discussions in PulPaper Congress in Helsinki, June 2010, it is obvious that traditional colony count methods cannot tell the truth about process problems.

These methods, originally developed for clinical microbiology, seem to have too high nutrient content. They cannot, therefore, select the "troublemakers" from the process samples. Bacteria like Gram-negative rods and Bacillus sp. are overestimated in these analyses but eg. filamentous bacteria cannot grow on common, commercial agar media.

Identification of bacteria can be important in some cases. Food poisoning species from the genuses Bacillus, Staphylococcus and Clostridia and hygiene indicators like coliforms, E.coli and Enterococci should be found in raw material control in the production of high hygiene products (LPB, other food-grade cartonboards and papers as well as tissue-type products). If not covered by other bacteria, they can be found with CC analyses. PCR also gives a good way to distinct them among other bacteria.

These methods cannot reveal some severe problems, however. Biofilm formation and comparative biocide testing are two types of investigations which cannot be performed with agar cultivations or molecular biology methods. They should be done either in machine trials or simulations. PMEU methods seem to be the best alternatives for rapid evaluation of biofilm formation and biocide testing today because they exclude all artefacts, caused by artificial growth medium (in colony counts) or too high selection of microorganisms (in PCR). CC's and PCR can be adopted to certain tests but when the subject of the study is to see, what happens in the real paper processes, simulation methods like PMEU shall be chosen.

The need of bacterial identification?

IM has discussed about alternative methods for the detection of hazardous or harmful bacteria with Dr. Elias Hakalehto.

It is most important to know the pathogens which will appear in patient samples. Clinical microbiologists shall know who are the enemies of the ill people: their metabolic capabilities, antibiotic resistence patterns etc. Their overall features are easy to find from literature or internet whenever the name of the species is known. This identification can be performed by selective cultivations on agar plates or in PMEU incubator, and further tests like microscopic examinations, API ID systems, immunological tests and/or PCR can be done to confirm the basic identification.

Paper mill is definitely another challenge for microbiologist. In some (relatively rare cases) the names of microorganisms are important to know: if the product shall have high hygiene quality (like LPB and other food-grade cartonboards) or questions about bioterrorism have been arisen (spore-forming Bacillus anthracis as an example). The occurrence of Legionella pneumophila is also a risk in the waste water treatment of paper industry today. Selective cultivations, either on plates or in PMEU, are the solid solutions for continuous microbiological control in those cases. PMEU is preferred because its speed (hours, compared to days with colony count analyses).

Papermakers shall focus more on the metabolic activities than the names of bacteria which they are living with in paper mills, however. Continuous inoculation of the paper production processes by contaminants, delivered with incoming lots of starches, mineral fillers, raw water, dry pulp etc. shall be controlled to avoid spoilage (amylolytic activity as an example), biofilm and slime growth, tastes and odours, spots and colours in the product etc. Because the wide range of bacterial species and their origin from the nature itself, clinical methods do not suit very well for this monitoring. There is no time to start labourous cultivations, pure cultures and identifications when the bacterial input continues day and night, "7/24". PMEU seems to be an excellent tool to check the basic features of process populations, their biocide resistence patterns included.

One important fact must also be taken into account. There are a lot of harmful microbes which actually cannot be cultivated on agar at all. One example are certain filamentous bacteria which may cause biofilm layers into the processes. They can be cultivated in some broths, however, but the usage of the original samples as the growth medium is the best way to detect them all. This can be done with ordinary mb laboratory equipment or with PMEU incubator.

Identification of bacterial species is still needed when the mapping of contamination routes into the processes is the subject of the study. IM will discuss about the microbiological mapping in his next posts.

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.