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.

Statistical methods in microbiology.

IM has discussed about the evaluation of novel microbiological methods with several professionals. His knowledge of statistical methods in microbiology bases on the lessons by Prof. Seppo Niemelä, who was (and still is) a well-known specialist in this not-so-well-known area of microbiology.

Testing of microbiological data is more complicated than similar analyses in chemistry. The main reason is the model of repeat distribution: chemistry follows the ordinary normal distribution but the colony counts of microbiological analyses are featured by the Poisson distribution. The reason for this difference is easy to understand: the count of molecules is overwhelming when compared to the limited count of colonies in microbiological cultivations. The dependence of variance on the mean of the data is another problem of colony count analyses, preventing the usage of parametric methods.

Luck enough, there are some non-parametric statistical analyses for Poisson-distributed data, helping the comparisons of means and trends of colony count results.

A novel problem seems to have arisen in microbiological evaluations since 70's. Many modern, automatized instruments of microbiology are not based on the measurements of chemical concentrations or counts of colonies but on specified metabolic activities of microbes. Examples of these analytical procedures are eg. measurements of impedance, turbidity, pH or CO2 production. Because these parameters are in a close connection to the growth rates - and to a new parameter, time - , their evaluations are very challenging procedures.

- More in next posts...

Annual Award "Forest Industry Trainer 2008" by AEL

Microbiology has regarded as a considerable area of paper industry support by Finnish training institute AEL a couple of days ago.

This award was definitely an issue of personal satisfaction for IM. The fact that the role of microbiological control of paper mills has been taken into consideration is the most important thing, however. Courses and lectures of P&P microbiology will focus the attention of personnel in this industry to the significant spares which are resulted by better maintenance of raw materials, run of the machines and quality of the products.

This award was given in the annual meeting of PI, Finnish Society of Paper Engineers. The forecasts of speakers in this meeting were optimistic ones and opinions seem to be pretty positive ones. Funding of several paper technology students also indicates these feelings. The final conclusion of this meeting can therefore be: P&P industry is not a "sunset branch" of global economy, no matter severe economical problems have arisen during last years. Paper and board is still needed worldwide, as well as new fiber-based bioproducts. Feelings after the meeting were therefore very good, indeed.

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.

How much do we understand?

As a microbiologist, IM knows that he can never understand microbial activities comprehensively. This is also known by all his fellows in the field of microbiology. 

How to predict the future steps of a microbial population? What are those factors which are important just on this very moment? Do we actually know them?

As an example: Ca2+ is needed for certain bacteria to produce spores. We still cannot, however, predict the sites of sporulation inside the paper machines because other essential triggers for sporulation may not be known by us.

This does not mean that we must rise our hands and thrust only on the old manners (like machine washings and boil-outs, set in annual calendars on the walls of control rooms). IM does not mean that tradition isn't important - in opposite! - but he would still like to have an optimistic view into the future of rapid microbiological process control. Novel ideas like Quality Control tool "HACCP", microbial activity estimations with "ON LINE ATP" and "PMEU" seem to be more and more well-known in P&P industry today.

Specialists and experts of technical issues begin also to understand the basic fact that it is basically impossible to understand all activities of microbial populations totally because the very complicated interactions of living and non-living components of the systems (which is definitely one of the facts in life which we must tolerate). But our "best guesses" can - and must - be derived from our basic knowledge of microbial metabolism, microbial ecology etc. When this expertise will be connected to laboratory and field experiments and experiences of paper process microbiology, the progress in the development towards better microbiological control of paper machine is secured.

- These ideas of paper industry microbiology seem to be very similar with those which must be understood and accepted in global economy today. No one really know what will happen to the infrastructure of the very complicated network of modern capitalism! Just to have ther very best tools for forecasts and planning!  

Better housekeeping for paper machines.

There has been definitely some improvement in the microbiological control of paper machines since late 1970's (when IM started his work for it). As a very short review, two issues have been highlighted because their positive effect on the hygiene and driveability of machines: application of oxidative agents as biocides and biofilm research. Rapid killing of microbes in strategic sites of processes with compounds like peracetic acid, chlorine dioxide and ozone have given very promising results when the overall load of microbes inside machines shall be dropped. Better understanding of the features of biofilms - essential changes of bacterial metabolism when they attach on surfaces from flowing water and start to release more and more living cells and spores back to the process  - also help to focus attention on them.

Poor interests to invest in better analytical control of microbial activities, in opposite, is surprising, however. Why do the mills not benefit the obvious progress of such control methods like ON LINE luminometry, rapid PMEU incubations etc.? Is it a question of lacking knowledge, lacking interest or the status of old-fashioned analytical methods, not suited to rapid QC/HACCP of paper and board processes? Is microbiology regarded as the unknown living creature on the surface of "Solaris" in the novel of S.Lem (the most excellent film by Andrei Tarkovsky recommended!)

IM has been relatively frustrated for this situation but tries to think optimistic: some small-scale simulations are already running today, and more and more people with technical background have been interested to know about tools to estimate and forecast microbiological events inside their machines. Happy to hear that they understand that with costs of only a couple machine rolls they could have improved microbiological control which can lead to remarkable spare of money when preventing unexpected machine stops or claims of poor product quality by customers.  

On the Threshold of a Dream: Application of rapid mb methods in paper industry

It seems that certain interest in novel, both rapid OFF LINE and automatized ON LINE  methods for the control of paper machine microbiology has arisen at least in Finland. The benefits of fast activities against spoilage of raw materials, biofilm and slime problems in processes as well as immediate corrections of biocide problems is understood in many mills.

What has prevent the progress of microbiological control in the mills until now?

First of all: lack of knowledge (ref. the picture above, drawn by my son in his early years). Bacteria, moulds and yeasts are something frightening for the majority of paper mill personnel. This opinion is false: learning basic facts of microbial ecology, environmental ecology and biotechnology helps to understand the activities of microbes. This kind of knowledge is served every year on trainig courses, intended in to present microbiology to laymen of paper industry.

Another claim: " We shall wash our machines several times per year - we then clean both chemical and microbiological problems away". False again: microbiological problems very often hide inside the process and lead frequently to acute problems which force to stop the production and clean the machine - and in worsest cases, lead to severe faults of products. 

We do not think that visiting our doctor a couple of times per year covers us against infectious diseases all the year, do we? Absolutely not - why then we thrust that infrequent boil-outs could prevent our machines against their "diseases"?

"Where we can catch information of novel control methods? We are not microbiologists?"

A good supply of paper industry microbiology is available nowadays, not only in scientific journals (J.Appl.Environ.Microbiology and J.IndMicrobiol.Biotech.  may be the best sources of P&P microbiology today),  but also on courses, blogs (like this and www.biotechtouch.blogspot.com) and elsewhere in Internet.

Focusing certain activity to be familiar with P&P microbiology isn't too hard task for those persons who are responsible for the technical and quality issues of paper and board production. It is definitely worth to do and to reserve time for it.

Back to the title of this post: great british rock group "The Moody Blues" published and album with the same title years ago. Progressive rock with jazz features (near to the heart of IM) then grew to a respective form of music all over the world on 1960's-1970's. I hope that we are now similarly stepping into a new era of control methods for paper industry microbiology today.

Shared responsibility for paper machine hygiene.

Current situation of process hygiene control in paper industry is relatively complicated.

No matter very effective control methods are already available (epifluorescence microscopy, ATP Assay, PCR, PMEU incubations) the roles of paper mills, biocide suppliers and raw material producers are not very clear.

Based on the experiences by Industrial Microbiologist since beginning of 80', it seems that paper mills prefer external services and this tendency is evergrowing today. Biocide suppliers have developed novel analyse methods which can be applied even at the mills, and many raw material suppliers know their response to deliver minerals, starches etc. with good hygienic quality for paper and board mills.

What should still be developed is the active role of paper mills themselves to control their machines. There is no more any need for expensive microbiological laboratories - in opposite, many methods could either be applied by wet end laboratories of the mills or be bought from external companies (ref. BIOTOUCH Service).

Wet end chemistry has already been controlled with ON LINE analyse units by certain institutes in Finland over years. ON LINE microbiological control is also coming soon, referring several current project proposals.

A combination of all parties - paper mills, biocide suppliers and raw material producers - would spare remarkable amount of money by optimizing biocide programs, by planning washing programs, by responsing immediately to hazardous situations and avoiding costs of returned products.

A paper machine can be regarded as an ECOSYSTEM, whose conditions are very stable because the strict adjustments of the process. It also acts as a BIOREACTOR with a continuous supply of nutrients and output of aged culture with its metabolites.


An essential feature of a paper machine process is its environmental stability (temperature, pH, redox potential, nutrients, stimulative and inhibitive factors etc.) which leads to the development of a selected, accommodated microbial populations in ecological "niches" all over the machine. The risk of microbial problems cannot usually be taken into account when optimizing these controls. This can lead to an active, waterborne population of various bacteria with total counts of even 1000 000 000 cfu/ml. Many essential raw materials of the paper production like starches, mineral fillers and coating paste components have also beneficial effects on the growth and activity of microorganisms.


These microbiological problems get ever worse by the formation of biofilms and slime layers. Aged biofilms are not only restricted sites of microbial growth but they also release bacteria and bacterial spores into the water circulations of the paper machine.


Storage tanks of raw materials can be regarded as batch fermentors where the typical growth steps LAG, LOG, STAT and KILL can be detected during extended storing periods. A paper machine itself acts like a continuous culture bioreactor or a chemostat, securing stable growt environment of the process-adapted microbial population.