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.

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.

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.

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.  

How to present novel mb methods to paper industry?

Novel methods of microbiology have been a most interesting topic for IM for decades. Beginning from 1970's, the value of ATPAssay and RR Test have been obvious, and both were adopted to paper industry on 1980's-1990's.

The increase of economical problems in paper industry are also very obvious today. IM has red articles and blogs about the reasons for this situation in this small, northern country with extensive forests: is it the problem of wood supply (= high Russian taxes for export and decreased activity by Finns themselves to sell their wood to the mills), the overcapacity of fine paper (other P&P products have no problems?) or both?

Back to the subject of my blog (after a short political survey, in which IM really has no expertice):

Paper companies are calling for tools to improve driveability of the machines, to catch better process control systems andto have lower number of product disqualification. Microbiological problems are still huge among all types of paper and board production, and QC methods like ON LINE ATP, BIOTOUCH Concept etc. could help significantly to lower the number of annual hours and days when the machines are stopped because microbiological problems.

But: how can we microbiologist publish these ideas? It seems that people, working for P&P industry, are more and more busy. How can we create a channel to deliver information about new methods to them?

Web-based marketing seems to be one challenging alternative to traditional paper articles, fairs etc. KK-Net (as an example) is doing a pioneer work to activate contacts between P&P industry and research institutes. This can be the way in future what we should follow. 

One must remember: Microbes are active day and night, no matter we may not are!

How to control the microbiological status of activated sludge?

There is one area of paper industry microbiology where active measures are needed to cultivate microbial flora: biological waste water treatment plants.

When focusing on activated sludge of aeration basin (anaerobic treatments will be discussed in another post), the roles of different microbes should first be understood.

The tasks of bacteria and protozoa - which are the beneficial microbial groups in the purification process - are different: bacteries are responsible for the transformations (mineralisation) of incoming organic compounds, Protozoa collects small particles (also bacteria) and acts as indicators of the activated sludge condition.

Bacteria shall also build up "flocs" which can settle down and be either returned backwards into the beginning of the purification process or totally removed from the plant. Significant amounts of nutrients will also be lead to the "sludge route" which is especially important for environmental reasons: no matter relatively low concentrations of nitrogen and phosphorus (compared with municipal waste water effluents), the volumes of P&P waste water effluents are huge.

Traditional parameters which are related to the microbiological status of activated sludge are C:P:R ratio, temperature, pH and oxygen concentration. In addition, the share of Protozoan indicator organisms are checked by microscopical methods.

ATP Assay is also a fast and reliable method to check the viability of activated sludge and control unfavourable sludge escape from secondary sedimentation basins (IM has written his second graduate work about this issue on 90's).

Incubations with PMEU equipment (FINNOFLAG Oy, Finland) have been very successful when the effects of low oxygen concentrations on the structure of flocs are the subject of the study. This method, in connection of bright field/dark field/phase contrast microscopy, gives also fast responses (even in hours) when the detrimental effects of toxic water fractions on activated sludge flora shall be checked.

There are many reasons for the bulking of activated sludge: nutrient supply, oxygen concentration, temperature etc. In all cases, a layer of activated sludge will rise on the surface of the basin and cannot be taken away from the water flow to sedimentation stages.

Measurements of redox potentials can give more information about the risks of anaerobic growth in biological waste water purification systems than sole oxygen concentration analyses(practical reslusts by IM). RO potential control may also work as a replacement/addition to traditional Respiratory Rate test, often used to check the viability of activated sludge.

Microbial activities continue also in sedimentation basins. IM has detected a significant rise of nitrogen concentration in certain basins in studies performed on 80's. The reason is obviously the activity of a coliform species, Klebsiella pneumoniae, which is able to fix nitrogen from atmosphere (nitrogen fixation). The routes of the pathogenic Legionella pneumophila in the purification process should also be investigated more deeply. Novel types of analytical methods to replace the traditional colony count methods (intended in the control of household water) shall be developed, however: L.pneumophila is very hard to "find" among other bacteria and fungi which are capable to grow extensively on selective nutrient media for Legionella.

ON LINE control has - until now - not got any role in HACCP of waste water treatment systems but promising technical solutions are already available for semi-continuous control of activated sludge microbiology.