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.