How to construct a rapid microbiological control for industrial processes and effluents?

How to construct an ON LINE analytical procedure to control the microbial growth in non-biotechnical processes like those in mining and paper manufacturing?

 First of all, it would be a good idea to map the microbiological problems of the process. They can be slime production by biofilm bacteria, biodeterioration of valuable raw materials or products, biocorrosion by sulfate reducing bacteria - these are the main subjects but, depending on the process in question, there are others, too. As an example, bacterial spores cause hazard for the hygiene of food packaging boards and papers, fermentative/anaerobic bacteria cause bad odours etc. HACCP (Hazard Analysis & Critical Control Points) examination, applied first in food industry over decades ago, helps a lot (more about HACCP in other post).

 Next step (and very important one) is the chose of the analytical method. There are several of them and many alternatives have been reviewed already in 1990 when "RAMI-90", Sixth International Congress on Rapid Methods and Automation in Microbiology and Immunology was held in Helsinki-Espoo, Finland, 7-10. June 1990. The German institute "Papiertechnische Stiftung" has also performed evaluations of rapid mibi methods on 80's. A summarizing article called "Microbiological Control of Pigments and Fillers" was presented by me in PIRA Symposium, Cambridge, England in 1997 and published in the series of  "The Fundamentals of Papermaking Materials". It describes the evaluations performed in Research Centre of ENSO Ltd. (currently STORA ENSO Ltd) and Helsinki University / Dept. Appl.Chem. and Microbiology). Some novel methods have appeared after this publication but, as we found in this research, ATP (Adenosine Triphosphate) analysis seems to be a valuable tool even today.

 The microbiological variables to be controlled must also be taken into account. If total growth is the main subject of the control, rapid biochemical reactions like ATP Assay or staining of the cells with fluorochromes like Acridine Orange are recommended. These two measurements are relatively simple and their results - values of light emission or fluorescence - can easily be handled as raw data, derived by optical measurements. RR (Respiratory Rate) Test is slightly more complicated because a certain incubation period of the sample is needed but it can also be automatized. Two drawbacks of this method are its low sensitivity and selectivity (only microbes with aerobic respiration can be detected). Whenever some special species (like coliforms) or groups (like SRO's = Sulfate Reducing Bacteria) are to be controlled, more time and money consuming methods like selective cultivations (in PMEU) or PCR are needed - and they are also very difficult to apply into an ON LINE control system.

 Collection and use of the data derived from the processes shall also be planned. Time series with certain transformations are usually most beneficial meters to show any kind of trend in the densities of free-floating (= non-biofilm) microbes. Growth rates of single-cell microbes in water environment will usually be presented after log transformation of microbial densities which gives a straight line in xy plots when the time scale is presented with equal intervals (semi-logarithmic plot). This means that the alarm threshold should be set wisely because the amount of microbial cells increases with a factor of ten in every time unit and the period of time which are needed for growth from 10 to 100 cfu/ml or 1 000 to 10 000 cfu/ml are equal  (if anything like lack of nutrients or any kind of inhibition doesn't prevent the growth). Statistical conclusions of biofilm and filamentous microbes will follow other guidelines and semi-logarithmic plot may not be the best framework to collect data. Solutions for the questions of statistical significance of rising or dropping microbial densities can be found in special textbooks like "Statistical Methods in Biology" by Norman T J Bailey (Edward Arnold, London). A Finnish  lesson about this subject, written by me for professional training centers, is also available by request.

Economical considerations will not be discussed deeper here because they are very much depending on the equipment and reagents. A very rough estimate for the price of one analysis is 10 - 30 € (which is, by the way, a relevant estimate for colony count analyses, too). When planning an automatized system, commercial instruments like luminometers may be applied to the system but they have to be modified to work  ON LINE (sampling, dosing of reagents, cleaning of the detectors etc.). The costs of control are very much depending on the time schedules and too frequent sampling shall be avoided.

Conclusion: an ON LINE microbiological control system can be constructed to collect time series of microbe density data and give early warnings of hazards whenever the target organisms and their critical growth sites are mapped (HACCP)  and the control method, depending of the specificity of organism(s), is chosen. Time series help to evaluate biocidic treatments, effects of the process conditions (temperature, pH, redox potential and so on), overall contamination of the process etc. and, finally,  threshold levels for the alarms, based on the control experiences, can be set.

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.

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.

PMEU as a tool for biofilm testing

PMEU Method was presented in PIRA Paper Industry Symposium, Barcelona, in October 2009. The basic PMEU model can be applied to diverse test types which help to construct biocide programs for the prevention of sessile and biofilm growth of bacteria in paper machines.

The picture above shows a typical test situation where process water sample, biocide and test coupon (made of steel) are installed in a PMEU syringe. Prevention of sessile growth can be monitored with ATP Assay, biofilm growth with UV Epifluorescence Microscopy.

In addition to the basic PMEU model, the novel PMEU Spectrion which measures the turbidity of all ten samples automatically, can be applied to any microbiological growth / growth prevention test of liquid or slurried samples from paper manufacturing processes. This device can handle also relatively turbid samples because it stops the mixing of the samples before turbidity measurement, allowing heavy particles (like mineral pigments) to sediment and enables therefore the measurement of bacterial cloudiness of the sample.

Competition between tube and colony count methods

All began with beef broth.

Dr. Louis Pasteur invented this method for the cultivation of diverse microbes. The famous "Swan Neck" trial was also performed with beef broth.

One of the first solid media for microbiological cultivations was the surface of a potato, presented by Dr. Robert Koch.

Colony counts began to be more and more favored by microbiologist because the colonies gave a chance to the immediate isolations of strains. The visual appearance of colonies on solid agars also help to identify the actors of the play, the species of a sample. Membrane filtration method also rise the popularity of colony count method, as well as the relatively good accuracy of colony count analyses, compared to the broth methods.

The role of the tube methods, however, has turned to be more important today. The limitations of the colony count method, correlated with the features of the samples (turbidity, toxic compounds etc.) and the slow growth (compared to the broth cultivation), are obvious. Testing of growth-affecting compounds like biocides are also easier and more reliable to perform in a solid media. When testing of those agents shall be done in the real environment (like the process water of a paper machine), the only alternative is the tube test. Detection of the response of stimulating and inhibiting agents can be done with various methods (photometry, colorimetry, turbidity, ATP Assay etc.) easily. Quantitative analyses of microbial counts can also be performed much faster with a (MPN) tube method than with the colony count method.

As a conclusion: colony count methods suit very well for purposes like the counting of CFU values as well as the selective cultivations of the total population to detect certain microbial groups. Testing of the effects of diverse growth factors (temperature, pH, biocidic and biostatic compounds etc.) should be performed with the tube methods, however. Growth on/in a solid medium does not correlate with the growth of the population in its original environment. Biofilm trials shall always be performed in liquids, never on solid media.

Various analytical tools have been developed for the measurement of the growth responses (pH, turbidity, impedance) automatically from the tubes and the most novel method, PMEU "mini-fermentor", gives the chance to perform all tests with the highest speed and - if needed - in the original samples to simulate the real growth environment of the microbial population. This method will be presented in PIRA Paper Conference, Barcelona, in next October.

A new PMEU application: quantitative MPN analyses of microbial counts

PMEU method is based on the rapid cultivations of several samples. The old idea to apply it in MPN (Most Probable Number) analyses has now proven to be correct: referring the Finnish Standard Book "SFS-KÄSIKIRJA 94: Mikrobiologiset vesitutkimusmenetelmät" (Methods for Microbiological Water Analysis) and discussions with specialists, PMEU can be used as an alternative, rapid method instead of the traditional technique, tube series in water bath or in an incubator. PMEU itself works as an incubator with a temperature deviation of < 0.1 oC.

A combination of 4 (levels of dilutions) * 5 (repeats) allows to follow the Finnish standard SFS 4447 (The Tube Method in Microbiological Water Analysis) as well as standards derived of it like SFS-EN ISO 9308-3 (for and coliforms) and SFS-EN ISO 7899-1 (for enterococci). Standards usually give MPN tables in the framework of 3*5 tubes (eg. for dilutions from 0 to 0.01) but PMEU gives an extra level (eg. 0 to 0.001) which covers a wider range of microbial counts. Samples with unknown levels of microbial densities are therefore easier to analyse correctly.

It seems that the leading status of membrane filtration has revised today. There are types of samples which are difficult or impossible to analyse with them (too much suspended solids etc.) and tube tests like MPN should be chosen. PMEU Tube Tests should be preferred also in situations where fast results (in hours, compared with days with colony count analyses) are needed.

The microbiological control of certain paper industry samples (pulp slurries,starches, minerals) are better to perform with tube methods. An example of the priority of the tube methods can be seen when samples with polymers should be analyzed: polymers tend to stuck membranes immiadely but do not prevent any analyses performed with tube methods. Rapid detection of harmful or hazardous bacteria can also be done faster with selective broths than in/on selective agars.

Applications of PMEU method for biofilm research and testing of biocides against biofilm growth

Paper machine biofilms have been studied already several years with PMEU method by IM. Test coupons, made of steel brands used in paper machines, have been installed inside PMEU syringaes and the growth has been observed with UV Epifluorescence Microscopy after a short incubation period (see picture above).

This technique has now been modified for ordinary light microscopes, too. Steel coupons have been replaced by specified glass slides and the Gram-stained biofilms can be observed with Bright Field Microscopy - no expensive epifluorescence microscopes are needed in this application.

This method will detect all biofilm-producing microbes and testing of biofilm-preventing biocides is also possible simultaneously. Primary attachers typically appear on the slides in just hours and mature biofilms are available in 12...24 hours. This application is very suitable for all areas of industry where the hygiene of surfaces is important. It can also be applied in every environmental research projects where the formation of biofilms in natural water environments is the subject of the study. Hygiene control of public swimming pools etc. also benefit of this method.

Sulphate Reducing Bacteria in paper industry.

SRB is an interesting group of bacteria which can use sulphate as an electron acceptor for the respiration. Despite the chances of certain other bacteria to use sulphate as the sulphur source for their S-containing cell components, the "real" sulphate reducers transform SO4(2-) to S(-2) in their energy metabolism and oxygen actually inhibits their growth - they are therefore obligate anaerobes. Certain yield of energy may be achieved through fermentation by SRB's but this type of metabolism is regarded as relatively insignificant one for them.

These bacteria have first detected in waste waters of sulphite pulp mills but modern paper machine processes can also induce their growth if certain sulphur-containing compounds are available.

In addition to H2S production (which is a hazardous gas), colour problems can arise because the metal sulphides. FeS is an indicator compound in the analytical detection of SRB's but also a harmful agent of discolorization of paper and paperboard. Last but not least, SRB's have been shown to be conneceted to a certain type of iron corrosion and it is all possible to find those problems still today whenever technical structures with poor steel quality and certain types of organic deposits on their surfaces are combined.

SRB's have also other, peculiar features like the tendency to follow non-exponential growth curve. They have been the subject of firm microbiological research only since the middle of 20th century because their need of anaerobiosis was not understood earlier.

FINNOFLAG Ltd. is currently developing a novel method for the sensitive detection of SRB's with PMEU method - more about this topic in next posts.

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.

"Top Three" microbiological problems of paper machines

Certain types of microbiological problems in paper mills seem to be acute all the time. Looking back to last months, this may be "Top Three" among them:

* Microbiological spoilage of raw materials. This is an everlasting hazard for mineral and starch slurries, and the reasons are very easy to understand: both raw materials mentioned may contain high densities of bacteria (mainly aerobic sporeformers and actinobacteria), slurries containing starch are very nutritive growth media for different microbial species and the very challenging biocidic treatments of slurries (especially mineral ones), when inaccurate, can lead to the total spoilage of them.

* Growth of biofilm and production of slime. This problem seems to be connected to poor washing and boil-out programs which leave rests of biofilm inside the machine and give growth time for it because too long running periods. The chose of ineffective biocide and/or its insufficient dosing can also stimulate the activity of these trouble-makers.

* Microbial growth in the broke system. Especially big machines with large broke towers suffer of this problem. If the basic biocide program is insufficient and the retention times inside the towers are too long, aerobic population tend to increase the number of its cells to the level of 10 000 000 cfu/g or even higher. Consumption of oxygen by respirating bacteria leads to anaerobic conditions, redox potential will be dropped and the growth conditions for both fermentative and anaerobic bacteria turns to be excellent. Drop of pH, slime and spore formation, smells and odours - even the production of H2S and H2 - will be found in such situations.

There are some measures to prevent these hazards. Growth period of microbial population shall be kept as short as possible, the control of waterborne and bioflim bacteria shall be as rapid and frequent as possible and the bioside programs, intended in killing of raw material, process water and biofilm bacteria shall be evaluated more frequently.

A realistic and accurate way to control both process water and biofilm growth, as well as to evaluate biocide programs, is now available. The Finnish company SAMPLION Ltd is manufacturing and selling "Portable Microbiological Enrichment Unit", a "mini-fermentor" for 10 simultaneous tests in controlled conditions, to detect the failures of biocide programs in only hours (watesr) or days (biofilms). Some results of PMEU's paper industry applications will be published in next Spring - coming back to refer them later.

Some wrong ideas about the overdosing of biocides will also rise up frequently among publicity. Basically it is not a question of only the cubic meters of biocides consumed, however, The chose of most effective biocides for different areas of processes towards different problems, the dosing of these compounds, their type of action and some other factors play a major role when building an effective biocide program for paper machine. Overdosing of biocides is a problem only in cases, when the program does not work, and leads to the loss of money and the rise of biocide concentration in paper machine effluents.

What's new?

Last weeks have been very active in IM's life.

Annual meeting of Finnish Paper Engineers's Union (Paperi-insinööriliitto) has given some new opinions about paper industry in our country. Optimistic views were mainly honest ones, and the title of the meeting indicated better views into the future: "There is light in the other end of the tunnel"!

Training of paper industry employees seems also to be continued. AEL (biggest professional training coalition in Finland) kept a course of paper industry sampling a couple of weeks ago in Lappeenranta, and AEL and its smaller "competitor", Finn-Fiber Oy (especially focused on paper industry issues) are planning new courses to be kept already in next summer and autumn. It is very interesting to join the planning groups of these companies and be with when finding current topics and speakers for seminars and lectures.

Main issue, of course, are the measures to spare money, and in this context, it means better drive of the machines. Better sampling and faster analyses of the processes are most important ways to prevent all kinds of process problems and product faults, and the role of PMEU (Portable Microbiological Enrichment Unit) seems to rise even more: a new coalition of companies called Samplion Oy has started the mearketing of this microbiological analyse tool, and it will be presented on Chembio Fair at the end of this month in Helsinki, Finland. We'll meet by the stand of Samplion Oy!

Negative issues should also be mentioned. One of them is the market situation of Russian short fiber: Carelian companies shall fulfill their task to collect certain amount of birch logs, no matter they cannot sell them to Finland because high taxes, set by Russian government. IM wishes good luck for the negotiations about this problem!

We are living interesting times - let's see what happens in the area of paper industry in next months!

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.

The need of paper machine biocides?

IM is wondering: are there any other sector of process industry, where microbes are allowed to grow like in paper industry?

Biotechnological processes, of course. But their populations are carefully selected and controlled. And they are employers of the company, not criminals trying to cause harm to the company.

Some significant changes in paper industry processes have taken place after the rise of active environmental care. Both the closure of water circulation and the cancelling of biocides having mercury as an active incredient are favorable for the environment, of course. But the microbial growth inside the paper machines has activated at the same time.

The beginning of neutral paper production led to an "ecocatastrophe" inside paper machines. The rise of pH value (as well as the rise of temperature, caused by the extended recirculation of water) was fatal for slow, acid-loving fungal growth. New mineral additives are an important source of certain types of bacteria, causing severe problems like production of slime, spoiling of the process compounds and hygiene faults of the products.

IM has published an article "Paper Machine: an Ecosystem and a Bioreactor" (INOCULA 1/2007. Helsinki, Finland). Many readers agree: paper machines offer ecological niches for bacteria, and the controlled environments of wet end circulations are very much similar with those of biotechnical processes, based on chemostatic fermentors.

A lot could be do to make paper machines more unfavorable growth environment for microbial contaminants. Very good results have been achieved in some projects where ecological aspects have been taken into account. These issues will be discussed later in this blog.

But the main question is: how much biocides we still need to control the microbial growth in all regions of a paper machine?

Incoming raw materials, sorry to say, may be very contaminated: the highest value of total count during IM's career has been over 100 000 000 cfu/g in a mineral slurry (which was fortunately replaced by a fresh lot by the supplier!). It is therefore obvious that a continuous control - both analytical and practical - is needed for starches, mineral pigments and other contaminated raw materials. This does not mean that all lots are spoiled: there are suppliers which know their response to deliver pure products to the mills but all kind of errors in biocidic pre-treatment, transport and storage of these products may happen.

Certain sites of paper machines also need biocidic treatments all the time. Chosing proper solutions for biocide programs (type of biocide, active compound, dosing sites, timing etc.) of a paper machine is a challenging tasks. In best cases, both the paper mill and biocide personnel are sitting down and discussing of the individual problems of the paper process hygiene.

When specified laboratory services, having tools like PMEU and biofilm microscopy, are included, the final result can be optimal one. Paper industry microbiologists can also help significantly by declaring the effects of process parameters on the growth of planktonic and biofilm bacteria.

The more competence is included, the better solution will be find.

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.