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.

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.

What will the next Spring bring to Finnish paper industry?

After a most busy period in May 2009, IM would like to write something (partly because his insomnia - a common symptom during light & bright Nordic summer nights) to the readers of his blog.

Several seminars for the rise of Nordic forest industry have arranged and will be arranged before Finland "will be closed" for summer holidays. - This is, by the way, a common feature of Finnish lifestyle, and easy to understand: you shall first work in dark and rain for months, the temperature drops well below 0 oC, and you should try to be active all through the winter. But when the summer comes, everybody will wake up again and "see a light at the end of a tunnel" (which was the title in the annual meeting of Finnish Paper Engineer's Association). And then, during the summer months, citizen tend to move to their summer cottages and villas to just rest and collect forces to tolerate the next winter..

Back to the topic:

There was a certain drop in the number of visitors, speakers and companies with exhibition desks in the annual Congress "BioChem" in Helsinki, 27.-29.5.2009. It was a pity to see that a lot of paper industry-associated companies did not arrive to show their products. The depression of global economy was very easy to sense. After all: there were still active participants left, eg. the novel company SAMPLION Ltd. which is now distributing PMEU system for all areas of microbiological control.

Positive attitudes were obvious also in the annual "Summer Summit" by AEL, the major education company for adult professionals, paper industry ones included.

What will happen next? A conference, discussing of future forecasts of forest industry, will be held in the middle of June in Jyväskylä, Finland. After that the annual PIRA congress will be held in Barcelona, Spain, in October.

If only the paper industry research will be activated again..It is, of course, a question of money, but, after all: to leave brilliant Finnish researchers without work is really a vast of excellence. It has be proven several times that the rebuilding of research groups after a depression is a much harder work than to start those activities from zero. It would therefore be better to keep such organisations in work over the bad periods.

IM is looking forward to the next Autumn! A most interesting thing to see what are the forecasts of Nordic P&P industry when the summer is over!