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)

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.

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 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.