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.