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.

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.

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.

On the Threshold of a Dream: Application of rapid mb methods in paper industry

It seems that certain interest in novel, both rapid OFF LINE and automatized ON LINE  methods for the control of paper machine microbiology has arisen at least in Finland. The benefits of fast activities against spoilage of raw materials, biofilm and slime problems in processes as well as immediate corrections of biocide problems is understood in many mills.

What has prevent the progress of microbiological control in the mills until now?

First of all: lack of knowledge (ref. the picture above, drawn by my son in his early years). Bacteria, moulds and yeasts are something frightening for the majority of paper mill personnel. This opinion is false: learning basic facts of microbial ecology, environmental ecology and biotechnology helps to understand the activities of microbes. This kind of knowledge is served every year on trainig courses, intended in to present microbiology to laymen of paper industry.

Another claim: " We shall wash our machines several times per year - we then clean both chemical and microbiological problems away". False again: microbiological problems very often hide inside the process and lead frequently to acute problems which force to stop the production and clean the machine - and in worsest cases, lead to severe faults of products. 

We do not think that visiting our doctor a couple of times per year covers us against infectious diseases all the year, do we? Absolutely not - why then we thrust that infrequent boil-outs could prevent our machines against their "diseases"?

"Where we can catch information of novel control methods? We are not microbiologists?"

A good supply of paper industry microbiology is available nowadays, not only in scientific journals (J.Appl.Environ.Microbiology and J.IndMicrobiol.Biotech.  may be the best sources of P&P microbiology today),  but also on courses, blogs (like this and www.biotechtouch.blogspot.com) and elsewhere in Internet.

Focusing certain activity to be familiar with P&P microbiology isn't too hard task for those persons who are responsible for the technical and quality issues of paper and board production. It is definitely worth to do and to reserve time for it.

Back to the title of this post: great british rock group "The Moody Blues" published and album with the same title years ago. Progressive rock with jazz features (near to the heart of IM) then grew to a respective form of music all over the world on 1960's-1970's. I hope that we are now similarly stepping into a new era of control methods for paper industry microbiology today.

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!