What will be happened in industrial-scale bioleaching?

What shall be taken into account when starting big-scale bioleaching? Some ideas:

1. A successive set of bioleaching pilots (with increasing size).

2. Transport of gases (O2, CO2).

3. Nutrients (P, N etc.).

4. Temperature.

5. Risk of re-building metal sulfides.

6. Microbial reduction of sulfate (see point 5.)

7. Chemical inhibition of sulfide-oxidizing metabolism.

8. Efficiency of Fe3+ to dissolute nickel.

9. Continuous control of microbial activities.

These are the basic issues. There may be more of them.

Coming back soon...

Bioleaching of Nickel - A Challenging Task

It seems that the retirement doesn't stop any person, dedicated to her/his work, to continue actual questions of her/his profession. My wife has worked as a nurse and still dreams about her work with schoolchildren...and the interests of mine are the questions of environmental and industrial microbiology. Can't help...

Bioleaching of metals from sulfide rocks is a most interesting thing! It happens in natural conditions on an area in Northern Europe where the rock contains sulfides of certain metals. Those bacteria which can perform this bioprocess are unique - they need bot oxygen and carbon dioxide as well as some other nutrients but no organic carbon source for energy at all. They are typically slow-growing microorganisms on the surface of the stone.

How to maintain an industrial-scale bioleaching process in mines where the conditions can be as different as in Northern Finland (cold, moist) and Australia (warm, dry)? This is the main question which I have studied by using scientific literature and results of laboratories (as far as they are public - what they not always are, indeed!), trying to compare different cases.

- More of the basics of bioleaching and cases in coming posts...keep following!

The Role of Microbes in Bio Boom - Part 2.

Bio Boom is here!

This is a great challenge for countries like Finland (a lot of forests)
and Central Europe (a lot of cattle farms).

What is common to both of them?

After the production of ethanol (and some other beneficial compounds 
(like lignosulfonate) from sulphite pulp process, very few biotechnical 
processes were applied to sulphate pulp processes.

Now it seems that ethanol can be an important product also in
forest industry.

And the cattle...?

Methane and hydrogen are important end-products of certain anaerobic
microbial processes. They were burned in previous times (I recall the
waste water treatment plant of Viikki, Helsinki, where no method to
collect these gases were available on 70's).

If we think the "end product of cows" we easily understand its value
in anaerobic gas production. Large farms in Central Europe are ready
 to apply these techniques for the production of burnable gases.

Third, a most important aspect too, may be easily forgotten: aerobic
waste water treatment (activated sludge process) in Finnish pulp &
paper mills. It has improved significantly the quality of such big rivers 
like Vuoksi and Kymijoki where big integrates of P&P industry
are situated.

- To be remembered: Bio Boom consists of many chemical and
technical innovations. These three issues, discussed above, are real
biotechnology. Biotechnology is a term for all processes which are
based on microbes (or their metabolic products like enzymes),
not only chemical reactions of non-living world. This means that
bioleaching of sulfide minerals is a real biotechnical process -
and makes it easier to understand the difficulties to carry it on.

The Role of Microbes in Biotech Boom, part 1.

The role of microbes in the energy production has been relatively small in older times. Burning of microbial biomass has not been a clever alternative because the tiny size of microbial cells (even billions of cells, 1 000 000 000, can be easily be suspended in one milliliter of water - all too slow to cultivate big lots of biomass) and their water content (drying would be too much energy-consuming procedure). Wood, in opposite, has been an excellent alternative, especially when the material has been dried enough to give positive net balance of energy.

The energy-containing metabolites of microbes, not the microbial cells themselves, have been the subject of research and development of energy production. Because the full-oxidized end-products of aerobic, oxygen-respiring micro-organisms, the fermentative microbes (like ethanol-producing yeasts) and anaerobic bacteria (like methane producers) have got a certain role in energy production. One big benefit of these applications are the non-expensive raw materials of metabolic routes like carbohydrates, manures and other stuff which has either been left from other processes or even regarded as a waste.

(- to be continued soon...)

Challenging R&D in forest industry: not only paper and timber products

"The largest forest industry centre in Europe is located in South-Eastern Finland", says the back cover text of this abstract booklet of Summer School 9.-10. September 2008 by Forest Industry Institute. Future forecasts, scenarios, innovations, specified methods - in an optimistic mood on those days. - But how today? Interesting to check the outcome of these ideas. - Looking forward...

Integrated Prevention of Microbial Growth in ecological niches of a paper mill

An average paper mill consists of several sites where waterborne and biofilm microorganisms can survive and grow. Different kind of populations can be found in different environments, e.g. wet mineral and starch slurries, coating pasts, white waters, pulps and brokes as well as all wet surfaces inside the paper production systems. The effective prevention of these sub-populations is depending of the biocide alternatives (storing, fast-acting or even sporicidic compounds) available. Their use should be combined with other measures, however: the prevention of their growth by process technology, production strategies, cleaning of the processes etc. - This combined activity, if clever planned, can the be called "Integrated Prevention of Microbial Growth".

The most beneficial tool for the planning of IPMG is the HACCP procedure which evaluates the potential microbiological hazards in every site and gives hints to choose the Critical Control Points in the total process.

But - what does the stimulation of microbial growth in a paper mill mean?

It shall be kept in mind when biological waste water treatment plant is the last step of the process water route from water source to recipient lake, river or sea. The growth conditions, in oppotie to those of manufacturing processes, should be as beneficial as possible to keep the growth rates of activated sludge or anaerobic treatment just on optimal level.

We have to understand the microbes and their needs when preventing or stimulating their growth activities. The count of microbes is only one variable - their responses to temperature, pH, redox potential and other growth factors, their metabolic features (e.g. sporulation-germination cycles), their tendencies to grow in water or surfaces) and other important factors should be taken into account when planning the programs for microbe prevention (inside the mill) or stimulation (biological waste water treatment).

- More about these issues in next posts...stay on the line.

(Schematic picture:  MENTU, J.V. 2001)

Pioneer of environmental microbiology!

I recall Dr. Howard E. Worne who was a pioneer in environmental microbiology, incl. bioleaching already on 70's. I met him in Finland when he was already over 70 years - very nice person, indeed.

The most frequent questions of industrial microbiology today?

Since the beginning of my blog, several years ago, I have collected some statistics about the questions which have been redirected from Internet search activities into my blog. The following "top three" is not actually statistically analyzed but still reviews my own opinion of the most important topics . The main issues, therefore, seem to be

* activated sludge
* hazardous bacteria (both pathogenic strains, like coliforms, in paper products and other specified pricrobiologyocess contaminants, like sulfate reducing bacteria, SRB's)
* prevention of biofilm and slime microorganisms

I have mainly focused on the effects of contaminating microbes in industrial processes. Biotechnological processes like production of beverages, antibiotics etc. have therefore excluded from my blog - the only exception is biological waste water treatment, however. I have also made a comparison between commercial bioreactors (fermentors) and paper machines because many similarities can be found when observing biotechnical productions  and process water systems of paper and board machines!

Despite my current focus on the environmental and mining microbiology, I still follow news concerning paper industry microbiology. I am also planning a mobile mibi service for small waste water purification units, agricultural facilities as well as for pulp & paper industry.

You are very welcome to follow my blog if these issues interest You!

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.

Novel method to produce biofuel - by E.coli!

As an (originally) environmental microbiologist, I was very pleased when reading the news, delivered by BBC web site, tonight:

A novel biotech method, based on the growth of coliform bacteria, has recently been tested in laboratory conditions by British scientists. This sounds very promising because the simple nutritive requirements and rapid growth of coliforms. E.coli has the world record of growth rates: it can duplicate in 20 minutes when growth environment is optimal. In addition, coliforms can use sugars, derived from mechanical pulp manufacture - suits also to Finland?

http://www.bbc.co.uk/news/science-environment-22253746

From paper industry to mining environment

After my career as paper industry microbiologist (1983 - 2009) I have totally reoriented towards environmental problems of mining industry today.

 Reasons for the closure of the paper microbiology development is the depression of this industrial area. No interest in rapid control methods can be found anymore. Positive attitudes still exist (esp. in Finnish innovation organisations) but "money rules" today. No economical support can be found for e.g. luminometric ON LINE methods from paper companies - and it is really a pity.

Short summary of my career in paper microbiology:

I actually started as environmental microbiologist in HU / Dep.of Microbiology, Helsinki, Viikki. I recall shortly the most interesting projects in the beginning of 80's: cyanobacteria, nitrogen fixation, Bacillus-based insecticides, soil microbiology, coliform research..and then I found myself in the projects of prof. Seppo Niemelä. We were able to perform species analyses of different waste water types and found the best indicators of paper industry effluents which were led to Lake Lohjanjärvi. Growth temperature ranges of most important indicator coliforms were also determined by Tmax method. Several scientific analyses were written and I started my doctoral thesis about environmental and clinical species of a certain coliform. - Thank you, Leena, for your help to detect plasmid-bound antibiotic resistances!

A big chance of my career happened in Autumn 1986 when I was accepted to work as the research microbiologist for Enso-Gutzeit Ltd. I had to leave my doctoral work (all documents still in my book shelf!) but I got a job where I learned everything about paper industry microbiology - from raw materials up to waste waters! There were countless persons who helped me to understand the world of paper making - I will thank them all  because the name list would be too long to be published.

After some reorientation in my private life I headed towards Jyväskylä in 2006 and had the great chance to work with late prof. Christian Oker-Blom (R.I.P., Chrisse!). We constructed several project proposals for paper industry with several Finnish and foreign companies but it seemed that there were no money left for this kind of projects in paper companies anymore. Some interesting projects of air quality etc. then follows (thank you, Markus!) in the last years of 00's.

The last post of mine was the key account director of Samplion Ltd. No matter my title, I was able to perform real laboratory evaluations in JAMK laboratory and field tests in paper and paper additive companies - interesting!

In addition to the posts mentioned, I have worked as a part-time teacher for professional teaching organisations (especially TL, AEL, POHTO), companies and universities of applied sciences (especially Imatra, Jyväskylä, Kotka).

Today, as formally retired, I am more and more activated in environmental microbiology (greetings to Helge and BIOTECHTOUCH!). Coming back to those years in Viikki - the circle is now closed. I wonder if I have to reconstruct my blog but let it wait...I'll try to inform my readers in Twitter and Facebook about my reorientation. Please follow the key word "Talvivaara"...!

A new era in microbiology: back to Pasteur's times?

(first published April 25th 2010 by Juha V. Mentu)

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.

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 machines themselves.

Monitoring of microbial growth and sporulation

pH, temperature, nutrients and biocides are usually observed when the risks of microbial growth and sporulation inside paper machines should be estimated. Fourth important factor, time, will be often ignored, no matter it plays a most significant role in paper process microbiology. The simple figure above shows two important parameters which reveal the microbiological status of the white water in an anonymous paper machine (my personal data ca. 2006): Colony Count (CC, indicating the count of vegetative bacterial cells per ml) and Spore Count (SC, indicating the count of bacterial spores per ml). It is obvious that this very sample contains relatively high amounts of nutrients to secure the rapid growth of bacteria (the growth rates of bacteria, carried by white water, are usually much lower but bacterial densities, in opposite, on a very high, stabile levels). What is especially important in this case is the rapid increase of bacterial spores - producers of process biofilms after their outgrowth and risks for product hygiene in their original form - which seems to take place after ca. 0,5 days storage time. This event takes place practically always when the flow of white water, pulp and broke have been stopped during the delays of constant machine operation. The hygiene status of the total process would then be very important to know - how much spoiled water and broke can be supplied from towers to the process, are there risks to have biodeteriorated raw materials from the storage tanks, is there any need to use shock dosing of biocides in some sites etc. - but it cannot be done by using CC, SC or any other cultivation technique which give analytical results only after several days' incubation periods. Long analytical delays in colony count analyses also totally prevent the efficient application of HACCP hygiene control procedures into paper machine environment. - These are the facts which motivate me personally to continue planning rapid, ON LINE process hygiene methods.

Microbiology Services for Paper Industry by BIOTECHTOUCH

BIOTECHTOUCH is a group of consulting specialists for process industry and environmental research. Our main competence areas are biocides, microbiology and toxicology. We have focused especially on the problems of paper industry (raw material, process and product hygiene as well as effects of waste waters on recipients). This is also my own area of activity today, after my departure from my previous post in SAMPLION Ltd.

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.