WO2004042082A1

WO2004042082A1 - A method for monitoring the presence of harmful microorganisms in paper industry

Info

Publication number: WO2004042082A1
Application number: PCT/FI2003/000839
Authority: WO
Inventors: German Jurgens; Marko Kolari; Frederick Rainey; Mirja Salkinoja-Salonen; Hanna Laatikainen; Päivi TAMMELA; Pia Vuorela; Pentti VÄÄTÄNEN
Original assignee: Kemira Oyj
Priority date: 2002-11-06
Filing date: 2003-11-06
Publication date: 2004-05-21
Also published as: AU2003276306A1; WO2004042082A9

Abstract

The invention is directed a method for monitoring harmful microorganisms in paper and board industry. In the present method the presence or absence of the harmful target microorganism is detected by in situ hybridization method using an oligonucleotide probe hybridizable with a region of a nucleic acid molecule of said microorganism. In particular, the microorganism may be selected from a group comprising the species Deinocossus geothermalis and the genera Meiothermus, Azospirillum, Burkholderia, Porhyrobacter, 'Rubellimicrobium' and Thermomonas.

Description

A method for monitoring the presence of harmful microorganisms in paper industry

Field of the Invention

The present invention relates to a method for monitoring the presence of harmful microorganisms in paper and board making industry and to the use of the method for determining the need for an anti-biofilm agent in the process. Furthermore, oligonucleotide probes as well as a kit assembly containing such probes are also provided.

Background Art

The paper and board making processes contain warm process waters (e.g. 45-60°C) that are rich in biodegradable nutrients and have a beneficial pH (e.g. pH 4-9) thus providing a good environment for the growth of microorganisms. The microbes in the process show biofilm-forming, i.e. surface-attached, growth and free-swimming, i.e. planktonic, growth. Several problems in the paper industry are caused by the biofilms, i.e. slime layers, which develop on the surfaces of the process equipments and can rip off from the surfaces. Biofouling of the machine surfaces and the loose biofilm/agglomerates can cause severe process disturbances: reduce water flow; block filters, wires, etc.; deteriorate the end-product quality, e.g. by causing holes or colored spots in the end product; or break the whole paper web. The biofilms are difficult to remove from the surfaces of the process equipment and often require the use of very strong chemicals.

For controlling the microbe growth e.g. biocides have been added to the process waters. Planktonic microbes have been efficiently controlled by the biocides; however, the use of biocides has not solved all biofilm problems in the paper or board machines. Several reasons exist for that, e.g. a wide variety of microbes are growing in the papermaking process and it is now also known that bacteria growing in a biofilm are generally more resistant to biocides than the planktonic microbes.

One of the present inventors, Kolari, M. has recently investigated the biofilm- formers in paper and board industry in Attachment Mechanisms and Properties of Bacterial Biofilms on Non-living Surfaces, Dissertationes Biocentri Viikki Universitatis Helsingiensis 12/2003, Ph.D. thesis, University of Helsinki, Finland. In the study biofilm-formers were identified and their interactions investigated with pure cultures, slime samples and isolated cultures using i.a. plating, staining and hybridization techniques. Accordingly, the processes were found to comprise different types of biofilm-forming microorganisms: primary biofilm-formers that are capable of adhering to clean surfaces and secondary biofilm-formers which then adhere to the primary biofilms. The primary biofilm-formers seem to be a prerequisite for successful surface colonization of several other microbes in paper and board machines. E.g. Deinococcus geothermalis is a primary biofilm-former and strains of several Bacillus species adhere to the primary biofilms of this bacterium. Kolari, M. et at. in Journal of Industrial Microbiology & Biotechnology (2003) 30, p. 225-238, describe more primary biofilm-forming bacteria. Amended with the newest, unpublished data, the relevant primary biofilm-forming microorganisms recognized thus far are: species Deinococcus geothermalis; genus Meiothermus, such as species M. silvanus or M. ruber; genus Azospirillum; genus Burkholderia, such as B. multivorans or B. cepacia; genus Porphyrobacter, such as P. cryptus; genus "Rubellimicrobium "; and genus Thermomonas, such as T. haemolytica or T. hydrothermalis. The primary biofilm-formers are typically moderately fhermophilic, some with maximum growth temperatures as high as 67°C, and most of the strains showing the fastest growth at temperatures of 45 to 55°C. To overcome the problem with the biofilms in paper industry was, however, not discussed in said thesis.

The efficient use of biocides would require an effective monitoring method. The typical monitoring method for the microbe growth in paper industry is the cultivation of a sample of process water in laboratory. Such cultivation methods are, however, time-consuming and complicated, i.e. they take several days and require i.a. the use of various growth media for isolation and identification of different types of microbes in a sample. Moreover, it is known that the microbe growth in laboratory is culture-medium-dependent, thus part of the microbes present in a sample are often unable to grow under the laboratory conditions. Furthermore, the monitoring is typically focused on the free-swimming microbes. Efficient methods for monitoring the effects of anti-biofilm agents on the biofilm-forming microbes have been lacking. Therefore, in the prior art biocides are conventionally used for non-selective inhibition of all microbes in the process, whereby high amounts of biocides are often required.

Identification and quantification methods based on the detection of nucleic acid sequences of organisms are well known in the field of biosciences. These methods include hybridization methods, wherein a probe of an oligonucleotide labelled with a detectable marker is developed which is complementary to a region of a DNA or RNA, e.g. rPvNA, molecule of the organism of interest. Such an in situ hybridization technique, also known as FISH (Fluorescence-In-Situ-Hybridization) is a well- established technique in the studies of microbial ecology (e.g. Amann, R. et al, Current Opinion in Biotechnology, 12, 2001, p.231-236). WO 0185340, WO 02103043 and WO 02102824 describe in situ hybridization method and/or a kit for certain industrial applications. However, the disclosed industrial applications relate to "pure" systems or systems with "specific microbes", wherein practically no microbe growth or only minor or specific microbe growth occurs, such as the use of in situ hybridization method in pharmaceutical industry or for detecting harmful microbes from drinking water or beer.

As stated above, in the context of slime formation in paper and board machines, it appears that the biofilm-formers are the most harmful microbes and their prevention would improve the functioning of the process machines and thus productivity of the process. However, there exists a need in the paper industry for more effective methods for monitoring the slime formation.

Object of the Invention

The object of the present invention is to provide a further method for the paper industry, which enables an efficient and timesaving means for monitoring the microbiological state of a paper or board making process, and, moreover, enables the recognition of harmful microbes before process failures emerge.

A further object of the invention is to provide a method, which can be used for specific monitoring of microbes that are relevant for biofouling in paper industry, to determine the need for an anti-biofilm agent in the process, and the effects of such an anti-biofilm agent.

Another object of the invention is to provide new oligonucleotide probes, which can be used for a reliable and fast monitoring of the harmful microbes, and for recognition of the causative bacteria in the case of slime outbreaks.

A still further object of the invention is to provide a kit assembly, which is very feasible for carrying out the method of the invention on the process site. Brief Description of the Drawings

Figure 1A and Figure IB present the epifluorescence microscope image and phase contrast light microscope image of the specific binding of the oligonucleotide probe shown in SEQ ID NO. 1 with a sample of the hybridization example 1.

Figure 2 presents epifluorescence microscope image of a paper machine biofilm of a red-gray slime sample after in situ hybridization with the probe shown in SEQ ID NO. 1 of the hybridization example 2.

Figure 3 presents epifluorescence microscope image of a paper machine biofilm of a red slime sample after in situ hybridization with the probe shown in SEQ ID NO. 1 of the hybridization example 2.

Figure 4 presents epifluorescence microscope image of a board machine biofilm on a sample support after in situ hybridization with the probe shown in SEQ ID NO. 1 of the hybridization example 2.

Figure 5 presents epifluorescence microscope image of Meiothermus ruber DSM1279 cells after in situ hybridization with the oligonucleotide probe shown in SEQ ID No. 14 of the hybridization example 3.

Detailed Description of the Invention

The invention is based on the idea that by controlling the growth of the biofilm- formers, and especially the primary biofilm-formers, during the paper or board making process the problems with slime formation could be prevented efficiently. Furthermore, the inventors found that by monitoring the biofilm-formers during the process the development of the more problematic slime in the whole biomass, which actually causes the biofouling, could be foreseen and/or predicted and preventive actions be started at an early stage, whereby the detrimental disturbances in the process could be decreased or fully avoided. However, the time consuming prior art methods used in paper industry are not suitable for monitoring quick changes in the process.

The present idea can unexpectedly be realized with in situ hybridization technique which was found to be surprisingly utilizable in a highly demanding microbial environment of the paper or board making process for monitoring selectively the presence of harmful microorganisms, especially the biofilm-formers and, among these, particularly the most harmful primary biofilm-formers. Moreover, it was found that the in situ hybridization method can be applied directly to samples taken from the process, i.e. without any prior cultivation or isolation steps and without any dilution/concentration steps, and that the targeted harmful microbe can be detected selectively in the presence of the versatile microbe population of the process.

The in situ hybridization method of the invention is very fast to carry out, i.e. samples taken from the process can be analyzed in three to four hours, whereby it is possible to take samples continuously from one or several locations of the process line, and to react quickly to the changes in the microbe growth within the process, i.e. to start of preventive countermeasures before process failures emerge, e.g. by dosing an anti-biofilm agent and by monitoring its effects by the said method.

The present method makes it possible to monitor more reliably and quickly the actual occurrence of the relevant microbe to be detected compared to the cultivation-based methods in current use. The method is also very sensitive, as single active cells of harmful microbes can be detected, and thereby the process personnel can be alerted before any visible process failures emerge.

The characterizing features of the invention are defined in the claims below.

"Paper or board making process" and "process line" as used herein covers the process lines of wet manufacturing processes for raw materials of paper and pulp industry, pulping process, the pulp drying process, the actual paper or board making process, and the equipment thereof including the paper and board machines. The paper and board machines are understood to comprise both the wet-end with wire section and the drying section.

Accordingly, the present invention provides a method for monitoring harmful microorganisms in paper and board making processes, wherein the presence or absence of the microorganism of interest in a sample is detected by in situ hybridization method using an oligonucleotide probe hybridizable with a region of a nucleic acid molecule of said microorganism.

Thus in the present method the oligonucleotide probe is hybridizable with a nucleotide sequence of a nucleic acid molecule present in the harmful microorganism desired to be detected (referred herein as "target microorganism" or "target microbe" or "microbe of interest"). Preferably, the probe is hybridizable with an agglomerate- and/or a biofilm-forming microorganism, more preferably with a primary biofilm-forming microorganism.

Due to the variation in the nucleic acid sequences, oligonucleotide probes can be designed for a chosen group of microbes or to a specific species. Preferably, the nucleotide sequence of the probe is targeted to a region of a nucleic acid molecule of the microbe of interest, which region is specific for that biofilm-forming genus or species. Furthermore, one or more probes can be used to hybridize with one or more target microbe species present in the sample, whereby each probe may be targeted to one microbe species.

Typically the oligonucleotide probe is hybridizable with a region of a DNA or RNA molecule, preferably of an RNA molecule, and more preferably of an rRNA molecule, of the harmful target microorganism. It is known that rRNA molecules are good target molecules for the in situ hybridization, as they exist in all living cells in detectable levels. Preferably the oligonucleotide probe hybridizes with a region, i.e. a nucleic acid sequence, present in the 16S or 23S rRNA molecule of the target microorganism. Preferably, the probe hybridizes with a region of the 16S rRNA of the desired microbe. It can also be said that the sequence of the probe is complementary to a specific region of the DNA or RNA molecule of the target microbe, whereby "complementary" means herein that the nucleotide sequence of the probe is complementary with at least part of the nucleotides within the length of the specific region of the DNA or RNA molecule of the target microbe, so that the probe hybridizes to the target region under suitable hybridization conditions. Thus, in manner known in the art, some degree of mismatches is allowed.

The sequence for the oligonucleotide probe specific to a target microorganism can be designed and prepared by methods well known in the field of bioscience using the nucleic acid sequences, e.g. rRNA gene sequences, of the target microorganism. The nucleic acid sequences of the target microorganisms may be found from the literature and public databases, or be identified e.g. by using the known PCR (polymerase chain reaction) and sequencing techniques. The probes designed can be compared for their specificity with the sequence information contained in the public databases such as the EMBL Nucleotide Sequence Database or the RDP II. For example the length of a probe can be between 12 and 1000 nucleotides.

The probe is typically provided with a detectable marker to enable the detection thereof after the hybridization. The marker is preferably attached to the 5 '-end of the sequence of the probe in a manner known in the art. Such markers may be commercially available and include a fluorescent marker, radioactive marker, enzymatically active group etc. One suitable marker is a fluorescent label, such as commercially available Alexa, Cy3, FITC or TRITC-labels.

In a preferred embodiment of the invention the method comprises the steps of:

(a) taking a sample from the process,

(b) fixing the sample to permeabilize the microbes present in the sample and, if needed, to immobilize the sample to a support,

(c) hybridizing the harmful target microorganism(s) present in the sample with one or more oligonucleotide probe(s) hybridizable therewith, preferably with a region of rRNA molecule of said target microorganism, and

(d) determining the presence or absence of said target microorganism by detecting the hybridized probe qualitatively and/or quantitatively.

In step (a) the sample can be taken from any part of the process line including the wet end part of the paper or board machines and the dry end of the process line. Furthermore the samples can be taken from any process media including process waters, pulp suspensions, circulating and waste waters, from any surfaces of the machines including liquid containing and splash areas of the machines, from wires, from filters, from raw materials, reagents, intermediate products and end products etc. The splash area in the process system includes splash areas of the paper or board machines, e.g. splash areas in the wire sections and process water containers. In a preferable embodiment the sample is from i) slimes collected from the machine surfaces, ii) raw materials of the process, iii) end products of the process, iv) process water, or v) biofilms grown on surface of sampler plates during immersion in the process water e.g. using a flow-through biofilm reactor.

The fresh sample is subjected to the support, e.g. an objective slide or a multi-well objective slide known in the art, for the fixing step.

If desired, e.g. in case that the probe is targeted to the rRNA of the microbe of interest, the sample, e.g. slime, can be activated prior the fixation step, e.g. in a suitable nutrient medium such as yeast extract, e.g. 30 min to 2 h, such as 1 h, between the ambient temperature and 65°C, preferably close to the temperature of the sampling site, e.g. at 35-65°C, e.g. at 40-60°C, to increase the rRNA content of the cells. The "fixing step" means the well-known permeabilization and/or immobilization of the microorganism present in the sample to a support to enable the hybridization of the probe with the desired DNA or RNA region of the target microbe. As an example e.g. dehydrating the sample by air-drying and/or treating with ethanol, could be mentioned.

In step (c) the hybridization is effected by incubating the fixed sample together with the oligonucleotide probe labeled with a detectable marker in conditions enabling the hybridization of the probe with the harmful target microbe, preferably with an agglomerate- and/or biofilm-forming microorganism, more preferably with a target primary biofilm-former, and then removing any unbound probe. It is generally known that the specificity of the probe to the target microbe is controlled by the hybridization conditions. Thus the hybridization can be effected in moderate or stringent conditions, preferably in stringent conditions to enable only the specific binding to the target RNA or DNA, such as rRNA, of the microorganism of interest. The stringency can be adjusted in the known manner e.g. by changing the concentration of the hybridization buffer, of NaCl and/or of formamide. Only as an example, e.g. 20 min to 3 h, preferably 2 h, at 40 to 55°C, preferably at 46°C, in a hybridization buffer such as 20 mM Tris-HCl (pH 7.2) including a detergent, preferably sodium dodecyl sulphate (SDS) 0.01%, sodium chloride, preferably 0.9 M, and formamide, preferably 20-45%, could be mentioned for achieving the stringent conditions.

Before the actual hybridization step (c) the fixed sample may optionally be pretreated with an unlabelled non-specific oligonucleotide probe(s) for blocking any unspecific binding sites in a manner known in the art.

The unbound probe is removed in a known manner by washing with a buffer solution. Only as an example washing with e.g. Tris-HCl buffer (pH 7.2) including SDS, e.g. 0.01% SDS, EDTA, e.g. 5 mM EDTA, and NaCl, e.g. 10-300 mM NaCl, at 40-60°C, e.g. at 48°C, for 20-30 min, could be mentioned.

Hybridized probe is then detected (d) qualitatively or quantitatively in a manner known in the art, e.g. a probe with a fluorescent label can be detected microscopically using an epifluorescence microscope. For quantitative determination the hybridized probes with the detectable marker can be separated in a known manner from the sample using e.g. solvents and quantified with a detection device such as a fluorometer. The standard in situ hybridization protocols, i.e. fixing, hybridizing and detection steps and the design of the probe, have been widely described in the handbooks in the field and the principles are within the skills of an artisan.

The inventors have further found that the present method can be used for detecting the relevant microbes from a biofilm formed in situ in the process on the surface of a sampler device and that the fixation and hybridization can unexpectedly be effected with said biofilm on the surface of said test sampler. Thus in a further preferred embodiment of the invention,

(a) the sample is a biofilm formed by biofilm-formers on a surface of a sampler support immersed in the process water or placed in a splash area within the process system for a period of time, e.g. for 4 h to 7 d, preferably 12 h to 48 h.

(b) microorganisms of the biofilm adhered to the surface of the sampler support do preferably not require fixing for immobilization on said support, only for permeabilization,

(c) a harmful target microbe, preferably a primary biofilm-former, present in the fixed biofilm is then hybridized on said sampler support with a labelled probe hybridizable therewith.

Preferably, (a) the sampler support is a test plate, e.g. a coupon or slide, preferably a slide of a stainless steel, and the biofilm is grown in situ in the process on the surface of the said test plate, (b) the fixing of the sample is effected on said plate, and (c) the in situ hybridization is performed on the surface of said plate. Preferably, the hybridization is effected in a disposable hybridization chamber attached, e.g. glued, on the test plate after the fixing step. Such disposable chambers are known from the bioscience and commercially available.

The sampler support device can be brought in the splash area or immersed in the liquid streams for enabling the biofilm-formers to form the biofilm on the surface of the sampler support. The sampler support may comprise means for fixing it e.g. on the surface of the paper making equipment, e.g. paper machine. Alternatively, the sample taking points of the process line may be provided with a mounting means, wherein the sampler device can be fixed for a period of time to enable the biofilm formation on the surface thereof. The time required for forming the biofilm on the surface of the device in the process depends on the place in the process line and can be chosen accordingly. E.g. when the device is in a wire water line of a neutral paper or board machine can some primary biofilm-formers be detected akeady after a six-hours exposure.

In a further preferred embodiment the oligonucleotide probe is hybridizable with a region of a DNA or RNA molecule, preferably of a rRNA molecule, of a microorganism selected from a group comprising species Deinococcus geothermalis, genus Meiothermus, particularly species M. silvanus or M. ruber, genus Azospirillum, genus Burkholderia, particularly B. multivorans or B. cepacia, genus Porphyrobacter, particularly P. cryptus, genus "Rubellimicrobium " and genus Thermomonas, particularly T. haemolytica or T. hydrothermalis.

Preferably, the probe is specific to the microorganism to be detected.

The invention further provides oligonucleotide probes, which were designed for the specific detection of the above listed microbes as given above, especially specific for a region of the rRNA molecule or its gene, especially of the 16S rRNA molecule, of said microbes. These probes provide an advantageous means for the method of the invention. Said probes include new specific oligonucleotide probes which comprise a nucleotide sequence selected from the listed sequences below, which sequences are also presented in the Sequence Listing as a part of this application with SEQ ID NO's indicated below. Accordingly, the probe of the invention comprises a nucleotide sequence selected: (from 5' — » 3' direction)

from nucleotide sequences, which are particularly suitable for the detection of Deinococcus geothermalis in a process sample, and preferably targeted for a region of the rRNA or its gene of said microbe:

-CCC ATC CGG GGC CTT TCG- which is shown in SEQ ID NO.l,

-TCT CCC CAT CCG GGG CCT TTC- shown in SEQ ID NO.2,

-CCC AAG CGG CCT CAG CCT TT- shown in SEQ ID NO.3,

-TCC CAC ATT AGC TCC CCT TTC G- shown in SEQ ID NO .4 ,

-CCT CAG CCT TTA CGT CGT TG- shown in SEQ ID NO.5,

-GCG GCA CGT TTC TCG CGT TG - shown in SEQ ID NO.6,

-CGG ACA GCA CAT CCC ACA TT- shown in SEQ ID NO.7,

-CTG CCA TGC ACT CCC ATG GTG- shown in SEQ ID NO.8, -CGG CCG TCA AGC TCC CGG TG- shown in SEQ ID N0.9,

-GCC TTT ACG TCG TTG CAT CT- shown in SEQ ID NO.10,

-AGT CCC CCT CGA GTC CCC GG- shown in SEQ ID NO.l 1,

-CAC CAC AGC CTA GAC ACC GG- shown in SEQ ID NO.12,

from nucleotide sequences, which are particularly suitable for the detection of genus Meiothermus, and preferably targeted for a region of the rRNA or its gene of said microbe:

-CTC CCA GCA CTG ATC GTT TAG- shown in SEQ ID NO.13,

-CCG CTA CTC CGG AAA TTC- shown in SEQ ID NO.14,

-CTA CTC CGG AAA TTC TGC- shown in SEQ ID NO.15,

-GAC CAG CTA CGC GTC GTC G- shown in SEQ ID NO.16,

-TCT CAG ACC AGC TAG GCG- shown in SEQ ID NO.17,

from nucleotide sequences, which are particularly suitable for the detection of Porphyrobacter cryptus, and preferably targeted for a region of the rRNA or its gene of said microbe:

-CGC CAC TAT CCC CGA AGG G- shown in SEQ ID NO.18,

-CCC TGA CTT AAA AGG CCG- shown in SEQ ID NO.19,

-AAG GGA TCG TTC GAC TTG- shown in SEQ ID NO.20,

from nucleotide sequences, which are particularly suitable for the detection of genus "Rubellimicrobium ", and preferably targeted for a region of the rRNA or its gene of said microbe:

-TGG GGA TAG ACC CTC TGT C- shown in SEQ ID NO.21,

-GCC ATG AAG ACG CCC GCT C- shown in SEQ ID N0.22,

-CCC GAA GCA GAA GGC ACG- shown in SEQ ID N0.23,

-CGC CGC TGA CGC CAT GAA- shown in SEQ ID NO.24, -TGC CTC CTC TCG CGA GGT- shown in SEQ ID N0.25,

-CAC GGG CAG TTC CCT CAG- shown in SEQ ID N0.26,

-CCC CCA GTT TCC CAA GGC- shown in SEQ ID NO.27,

or a functional variant thereof.

In the method of the invention one or more probes, preferably one or more new probes as specified above, can be used.

"Functional variant thereof means that the probe sequence may have variations compared to the nucleic acid sequence listed above, but the variant probe is still capable to hybridize to the target microbe in suitable hybridization conditions. E.g. the sequence of the probe may have 60 % or more, e.g. 80 % or 95 %, similarity with the corresponding above listed sequence. Thus mismatches are allowed in certain degree as mentioned above for the term "complementary".

"Functional variant thereof means also peptide nucleic acid molecules that could substitute for the oligonucleotides.

The method of the invention may preferably be used for monitoring the presence of the harmful microorganisms, especially the primary biofilm-forming microorgamsms, in the paper and board machines, in pulp-drying machines, and in the wet manufacturing processes for raw materials of paper and pulp industry.

In one preferable embodiment of the method of the invention, the presence of the biofilm-forming microorganisms, especially the primary biofilm-formers, is monitored for determining the need of an anti-biofilm agent in the paper or board making process and/or for determining the effects of such an anti-biofilm agent.

The invention further provides a method for inhibiting the formation of agglomerates and/or a biofilm by thermophilic microbes capable of adhering to the surfaces of the paper and board machines and/or for removing such agglomerates and/or biofilms from said surfaces, wherein at least one anti-biofilm agent is added in an amount effective against said microbes to the circulation water of said paper or board making machines, whereby the need of said anti-biofilm agent is monitored with the detection method of the invention.

With the accurate, sensitive and fast detection method the process can be monitored continuously, whereby the need of an anti-biofilm agent, the timing of the addition and/or of any further addition as well as the doses of the anti-biofilm agent can be optimized over to the prior art.

Anti-biofilm agent means herein generally any substance suitable to paper industry for the inhibition or prevention of the growth of harmful microbes, preferably of biofilm- and agglomerates-forming microorganisms, and, particularly, for the inhibition or prevention of the formation of a biofilm and/or removal of the formed biofilm from the surfaces of the process equipment. Such substances include any compounds, mixtures of compounds and extracts, i.a. chemicals and biocides conventionally used in the paper industry, such as 2,2-dibromo-3- nitrilopropionamide (DBNPA) and methylene bisthiocyanate (MBT), or effective agents of plant origin, compounds and extracts, such as those mentioned below.

Preferably, the anti-biofilm agent used for inhibiting the biofilm-formation in the present method is a pure substance isolated from a plant or a plant extract, or a mixture thereof.

Herein the terms "agent originating from a plant", "pure compound" and "plant extract" mean a natural agent isolated from a plant or a synthetic equivalent or derivative thereof, which has an ability to inhibit the formation of the biofilm by thermophilic microbes and/or to remove such biofilms from a surface. The reduction achieved in the biofilm-formation should be at least 50 %, preferably at least 70 % and more preferably at least 90 %. The agents of plant origin can reduce the adhering of the biofilm-formers, some of them up to 90 % or more.

For instance the plant extracts can prevent effectively, 91-99.7 %, the agglomerate forming D. geothermalis of adhering on the surface of the paper machine steel (AISI 316). The prevention achieved e.g. with betuline and flavonols varies between 84-99.95 % depending on the used agent.

Accordingly, the "agents originating from the plant" can be used as raw extracts or as more effective components isolated from said extracts. The "agent originating from a plant" or "pure compound" or "plant extract" effective against biofilm- formation may originate e.g. from Japanese rose (Rosa rugosa), rosebay willow herb (Epilobium angustifolium), meadowsweet (Filipendula ulmarid), or salvia (Salvia officinalis), and against agglomeration e.g. from deciduous trees, such as rowan tree (Sorbus sp.), birch (Betula sp.), maple (Acer sp.) and willow (Salix sp.), bark of a conifer, such as pine bark, a decayed conifer, such as spruce branches, or dwarf shrubs or wooded field vegetation, such as lingnonberry and blueberry. The extract can be obtained by extracting the plant or a part of it with a solvent or a mixture of solvents. One preferable solvent is methanol. Other suitable solvents for the extraction include cold water, acetone, ethanol, hexane and chloroform.

Examples of the "agent originating from a plant", e.g. "pure compound" or "extract", against biofilm-formation are phenolic compounds, such as an ester of a phenolic acid. A preferable ester of a phenolic acid is an alkyl ester of a gallic acid, preferably octyl gallate or lauryl gallate or a mixture thereof, and against agglomeration betuline and flavonols, such as pentahydroxyflavone and trihydroxyflavone, or a mixture thereof.

The "agents originating from a plant", such as "pure compound" or "extract", may be added e.g. to circulating waters of a paper or board machine to a product concentration of 1-1000 ppm, preferably 5-200 ppm, and more preferably 10- 100 ppm, calculated from the dry weight of the "agent originating from a plant".

The invention further provides an assembly kit for monitoring the presence of agglomerate- and/or biofilm-forming microorganisms in a paper or board making process. Said kit comprises one or more oligonucleotide probes hybridizable with primary biofilm-forming microorganism(s) occurring in paper and board industry and labelled with a detectable marker.

According to a preferable embodiment the assembly kit comprises:

- a support for receiving the sample and for fixing the sample on the support,

- reagents for fixing the sample,

- hybridizing buffer solution containing the oligonucleotide probe(s) hybridizable with primary biofilm-forming microorganism(s) occurring in paper and board industry and labelled with a detectable marker,

- washing buffer solution, and optionally

- unlabelled non-specific oligonucleotide(s) for blocking any unspecific binding sites

More preferably, the assembly kit according to the invention comprises one or more new specific oligonucleotide probe(s) as defined above. The reagents for fixing the sample and the hybridizing and washing buffers are in general known to a skilled person and may be chosen to be suitable for the present microorganisms and probe(s). Suitable examples are described above and can be generalized from the examples below.

In a preferred embodiment of the assembly kit, the support for receiving the sample is a sampler support as defined above, e.g. a test plate, to be placed in the process for enabling the biofilm-formers to form a biofilm on the surface thereof in situ in the process, to be used as a support for the fixation, i.e. permeabilization, of the microorganisms present in the biofilm formed on the surface thereof and for the hybridization of the oligonucleotide probe(s) with the target microorganism(s) present in the biofilm formed on the surface thereof, and optionally for the detection step. Furthermore, in this embodiment the kit may further comprises miniature hybridization chamber(s), e.g. those known in the field of bioscience and commercially available, which can be attached, e.g. glued, on the support for retaining the hybridization buffer on the support, e.g. on the test plate.

If desired the assembly kit can also be provided with a detection device for the label used for the probe. E.g. for a fluorescent label an epifluorescent microscope can be used.

In a still further embodiment, the hybridization method and the assembly kit of the invention can be used for detecting the efficacy of an anti-biofilm agent against biofilm-formers in a process, whereby the specific hybridization for the biofilm- formers is performed after the sample has been exposed to an anti-biofilm agent.

Examples

Experiments conducted

Samples in the experiments were 1) bacterial pure cultures in active growth phase in liquid growth media, 2) slime samples freshly collected from surfaces of paper and board machines, and 3) biofilms grown on stainless steel test plates which had been immersed in the process water of paper or board machine for 2 d.

The used reagents and starting material, unless otherwise stated, are cornmercially available.

Sample pretreatment: Bacterial cultures (a 8 μl) or slime samples were immobilized by air-drying to the surface of microscopy slides (Teflon printed glass slides, 10- well, coated with gelatin) and then dehydrated by successive passages through 50%, 80%, 96% and abs. ethanol (3 min each). Biofilms grown on stainless steel test plates were treated similarly, except that miniature hybridization chambers (Secure- Seal™ hybridization chamber, Molecular Probes, Eugene, OR, USA) were glued on the stainless steel test plates after the dehydration step. These disposable plastic chambers were used in order to prevent the hybridization buffer from running off the steel surface during hybridization.

In situ hybridization: Pre-warmed hybridization buffer (+46°C; 20 mM Tris-HCl pH 7.2, 0.01% SDS, 0.9 M NaCl and X¹ formamide) containing 1.1 μM of Cy3- labeled probe was pipetted (10 μl) in each well of the microscopy slide / (40 μl) in each hybridization chamber glued on the stainless steel surface. The cells were hybridized for 2 h at 46°C. After this the chambers were removed and discarded, and the steel plates /microscopy slides were shortly rinsed with warm (46°C) hybridization buffer without the probe and transferred to the washing buffer (20 mM Tris-HCl pH 7.2, 0.01% SDS, 5 mM EDTA and ² mM NaCl) at 48°C for 20 min. The slides were rinsed with distilled water and air-dried. The results were read under an epifluorescence microscope using a lOOx oil immersion objective.

Successful in situ hybridization requires stringent conditions that allow binding of the probe to the completely matching target rRNA sequences only. This is achieved by adjusting the formamide concentration of the hybridization buffer (X¹) and the NaCl concentration of the washing buffer (X ). The stringent conditions for each probe were experimentally screened.

Example 1. Specific labeling of the primary biofilm-former Deinococcus geothermalis

An oligonucleotide probe shown in SEQ ID NO.l (5'-CCC ATC CGG GGC CTT TCG- 3') was designed for specific detection of the primary biofilm-former Deinococcus geothermalis. The sequence of SEQ ID NO.l -probe was compared to the prokaryotic 16S rRNA gene sequences (ca. 72000 sequences at 26.6.2003) in the RDP-II database (Ribosomal Database Project at http://rdp.cme.msu.edu/html/). Only four strains with completely matching target 16S rRNA sequences were found, covering all strains representing the species D. geothermalis in the database: strains AG-3a^T, AG-5a and RSPS-2a from hot springs and E50053 from paper machine. The probe was separated from the next nearest hit in the RDP database (Brochothrix thermosphacta ATCC11509^T) by three mismatching bases indicating that SEQ ID No.l -probe is specific for D. geothermalis. Figure 1 shows specific binding of the probe shown in SEQ ID NO.l to the cells of primary biofilm-former Deinococcus geothermalis. Epifluorescence microscope image (Figure 1A) and phase contrast light microscope image (Figure IB) taken from the same spot of the specimen after in situ hybridization with SEQ ID NO.l - probe (X¹ = 40% and ² = 20 mM). The specimen was a mixture of broth cultures of eight different species: D. geothermalis E50053, Bacillus amyloliquefaciens TSP55, B. licheniformis D50141, B. megaterium HAMBI696, B. pumilus TSP66, D. radiodurans DSM20539 , Micrococcus luteus HAMBI1399 and Staphylococcus aureus HAMBI66. The epifluorescence microscope image in figure 1A shows that only cells of D. geothermalis E50053 were labeled by the fluorescently tagged SEQ ID NO.l -probe, whereas the phase contrast image in Figure IB shows cells of all eight species in the specimen.

Figure 1 demonstrates that cells of the primary biofilm-forming species D. geothermalis can be specifically hybridized in situ with the probe shown in SEQ ID NO.l and thereafter microscopically detected despite of the presence of other bacteria in the same specimen.

Example 2. Specific detection of the primary biofilm-former Deinococcus geothermalis in paper-machine biofilms

The presence of D. geothermalis in paper and board machine slimes was examined by in situ hybridization and the probe shown in SEQ ID NO.l.

Figure 2 shows epifluorescence microscope image of a paper machine biofilm after in situ hybridization with probe shown in SEQ ID NO.l (X¹ = 40% and X² = 20 mM). The red-gray slime was sampled from the vicinity of the first press cylinder of a magazine paper machine (52°C, pH 7). Image shows a brightly fluorescent microcolony (arrow) of coccoidal cells that were labeled with SEQ ID NO.l -probe indicating that they are D. geothermalis cells.

Figure 3 shows epifluorescence microscope image of a paper machine biofilm after in situ hybridization with the SEQ ID NO.l -probe (X¹ = 40% and X² = 80 mM). The red slime was sampled from the splash area of wire section of a newsprint machine (52°C, pH 5). Almost all cells visible in the image were labeled by the SEQ ID NO.l -probe (intensive orange fluorescence) indicating that they were D. geothermalis cells. Based on microscopic cell counting D. geothermalis constituted ca. 10% of the all bacteria in this particular slime specimen. Figure 4 shows epifluorescence microscope image of a board machine biofilm after in situ hybridization with the SEQ ID NO.l -probe (X¹ = 40% and X² = 20 mM). The biofilm was formed on a stainless steel test plate during a two-day immersion in the cloudy-filtrate container (52°C, pH 7). Image shows only a few cells labeled with the SEQ ID NO.l -probe. Based on microscopic cell counting D. geothermalis constituted less than 1% of the all bacteria in this particular slime specimen indicating that in this site of the process the major primary biofilm-former was other species than D. geothermalis.

Figures 2, 3 and 4 demonstrate that the presence of the biofilm-forming D. geothermalis in process samples can be monitored using in situ hybridization and specific probes. This approach is faster than the traditional cultivation-based approaches. In the two paper machine slime specimens (Figures 2 and 3) the cells of D. geothermalis were abundant, whereas in the board machine specimen (Figure 4) this adverse bacterium was present only in low amounts.

Example 3. Specific labeling of the primary biofilm-forming bacteria of genus Meiothermus

An oligonucleotide probe shown in SEQ ID NO.14 (5' -CCG CTA CTC CGG AAA TTC- 3') was designed for specific detection of the primary biofilm-forming bacteria of the genus Meiothermus. The sequence of SEQ ID NO.14 -probe was compared to the prokaryotic 16S rRNA gene sequences in the RDP-II database. Twelve completely matching target 16S rRNA sequences were found, all originating from strains belonging to the genus Meiothermus. The probe was separated from the two next nearest hits in the RDP database (Lactobacillus vaginalis and Paenibacillus pabulϊ) by four mismatching bases indicating that SEQ ID NO.14 -probe is specific for bacteria of the genus Meiothermus.

Figure 5 shows epifluorescence microscope image of Meiothermus ruber DSM1279^T cells after in situ hybridization with SEQ ID NO.14 -probe (X¹ = 15% and = 180 mM). The hybridized cells show intense orange fluorescence.

Figure 5 demonstrates that primary biofilm-forming bacteria of the genus Meiothermus can be specifically hybridized with the SEQ ID NO.14 -probe.

Claims

1. A method for monitoring harmful microorganisms in paper and board industry, characterized in that the presence or absence of the harmful target microorganism is detected by in situ hybridization method using an oligonucleotide probe hybridizable with a region of a nucleic acid molecule of said microorganism.

2. The method according to claim 1, characterized in that the target microorganism is an agglomerate- and/or a biofilm-fomiing microorganism.

3. The method according to claim 2, characterized in that said microorganism is a primary biofilm-forming microorganism.

4. The method according to any of the preceding claims, characterized in that the oligonucleotide probe is hybridizable with a region of a DNA or RNA molecule, preferably of an rRNA molecule, such as a 16S rRNA molecule, of the microorganism.

5. The method according to any of the preceding claims, characterized in that the detection method comprises the steps of:

(a) taking a sample from the process,

(b) fixing the microorganisms present in the sample to a support,

(c) hybridizing the target biofilm-forming microorganism present in the sample with an oligonucleotide probe hybridizable thereto, and

6. The method according to claim 5, characterized in that (c) the hybridization is effected by incubating the fixed sample together with one or more oligonucleotide probes labeled with a detectable marker in conditions enabling the hybridization of the probe(s) with the target microbe(s), preferably with a target primary biofilm- formers), and then removing any unbound probe.

7. The method according to claim 5 or 6, characterized in that (a) the sample is taken from i) slimes collected from the machine surfaces, ii) raw materials of the process, iii) end products of the process, iv) process water, or v) biofilms grown on surface of test plates during immersion in the process water e.g. using a flow- through biofilm reactor.

8. The method according to any of the preceding claims 5-7, characterized in that

(a) the sample is a biofilm formed in situ in the process on the surface of a sampler support immersed in the process water or placed in a splash area of the process system for a period of time,

(b) microorganisms present in the biofilm on the surface of the sampler support are fixed on said support,

(c) the harmful target microorganism, preferably the primary biofilm-former, present in the fixed biofilm is hybridized on said support with a labelled probe hybridizable therewith.

9. The method according to any of the preceding claim 8, characterized in that (a) the sampler support is a test plate and the biofilm is grown on the surface of the test plate, (b) the biofilm is fixed on the plate to permeabilize the microorganisms present on the biofilm, and (c) the in situ hybridization is performed on the test plate, preferably in a disposable hybridization chamber attached on the test plate.

10. The method according to any of the preceding claims, characterized in that the oligonucleotide probe is hybridizable with a region of a DNA or RNA molecule, preferably of a rRNA molecule, of a microorganism selected from a group comprising species Deinococcus geothermalis, genus Meiothermus, particularly species M. silvanus or M. ruber, genus Azospirillum, genus Burkholderia, particularly B. multivorans or B. cepacia, genus Porphyrobacter, particularly P. cryptus, genus "Rubellimicrobium " and genus Thermomonas, particularly T. haemolytica or T. hydr other mails.

11. The method according to any of the preceding claims, characterized in that the probe is specific to the target microorganism.

12. The method according to any of the preceding claims, characterized in that the probe comprises an oligonucleotide sequence selected from: (from 5' — » 3' direction) -CCC ATC CGG GGC CTT TCG- shown in SEQ ID NO.l,

-TCT CCC CAT CCG GGG CCT TTC- shown in SEQ ID NO.2,

-CCC AAG CGG CCT CAG CCT TT- shown in SEQ ID N0.3,

-TCC CAC ATT AGC TCC CCT TTC G- shown in SEQ ID NO.4,

-CCT CAG CCT TTA CGT CGT TG- shown in SEQ ID NO.5,

-GCG GCA CGT TTC TCG CGT TG - shown in SEQ ID NO.6,

-CGG ACA GCA CAT CCC ACA TT- shown in SEQ ID NO.7,

-CTG CCA TGC ACT CCC ATG GTG- shown in SEQ ID NO.8,

-CGG CCG TCA AGC TCC CGG TG- shown in SEQ ID NO.9,

-GCC TTT ACG TCG TTG CAT CT- shown in SEQ ID NO.10,

-AGT CCC CCT CGA GTC CCC GG- shown in SEQ ID NO.l 1,

-CAC CAC AGC CTA GAC ACC GG- shown in SEQ ID NO.12,

-CTC CCA GCA CTG ATC GTT TAG- shown in SEQ ID NO.13,

-CCG CTA CTC CGG AAA TTC- shown in SEQ ID NO.14,

-CTA CTC CGG AAA TTC TGC- shown in SEQ ID NO.15,

-GAC CAG CTA CGC GTC GTC G- shown in SEQ ID NO.16,

-TCT CAG ACC AGC TAC GCG- shown in SEQ ID NO.17,

-CGC CAC TAT CCC CGA AGG G- shown in SEQ ID NO.18,

-CCC TGA CTT AAA AGG CCG- shown in SEQ ID NO.19,

-AAG GGA TCG TTC GAC TTG- shown in SEQ ID NO.20,

-TGG GGA TAG ACC CTC TGT C- shown in SEQ ID NO.21,

-GCC ATG AAG ACG CCC GCT C- shown in SEQ ID N0.22,

-CCC GAA GCA GAA GGC ACG- shown in SEQ ID NO.23, -CGC CGC TGA CGC CAT GAA- shown in SEQ ID N0.24,

-TGC CTC CTC TCG CGA GGT- shown in SEQ ID N0.25,

-CAC GGG CAG TTC CCT CAG- shown in SEQ ID N0.26,

-CCC CCA GTT TCC CAA GGC- shown in SEQ ID N0.27,

or a functional variant thereof.

13. The method according to any of the preceding claims, characterized in that the method is for monitoring the presence of the harmful microorganisms, especially the primary biofilm-forming microorganisms, in the paper and board machines, in pulp-drying machines, and in the wet manufacturing processes for raw materials of paper and pulp industry.

14. The method according to any of the preceding claims, characterized in that the presence of the biofilm-forming microorganisms, especially the primary biofilm-formers, is monitored for determining the need of an anti-biofilm agent in the paper or board making process.

15. A method for inhibiting the formation of agglomerates and/or a biofilm by thermophilic microbes capable of adhering on the surfaces of the paper and board making machines and/or for removing such agglomerates and/or biofilms from said surfaces, characterized in that at least one anti-biofilm agent is added in an amount effective against said microbes to the circulation water of said paper or board making machines, and in that the need and the performance of said anti-biofilm agent is monitored with the method of any of the preceding claims 1-14.

16. The method according to claim 15, characterized in that the anti-biofilm agent is a plant extract or a pure substance isolated from a plant, or a mixture thereof.

17. An oligonucleotide probe comprising a nucleic acid sequence selected from the nucleotide sequences: (from 5' — > 3' direction)

-CCC ATC CGG GGC CTT TCG- shown in SEQ ID NO.l,

-TCT CCC CAT CCG GGG CCT TTC- shown in SEQ ID NO.2,

-CCC AAG CGG CCT CAG CCT TT- shown in SEQ ID NO.3, -TCC CAC ATT AGC TCC CCT TTC G- shown in SEQ ID NO.4,

-CCT CAG CCT TTA CGT CGT TG- shown in SEQ ID N0.5,

-GCG GCA CGT TTC TCG CGT TG - shown in SEQ ID N0.6,

-CGG ACA GCA CAT CCC ACA TT- shown in SEQ ID N0.7,

-CTG CCA TGC ACT CCC ATG GTG- shown in SEQ ID NO.8,

-CGG CCG TCA AGC TCC CGG TG- shown in SEQ ID N0.9,

-GCC TTT ACG TCG TTG CAT CT- shown in SEQ ID NO.10,

-AGT CCC CCT CGA GTC CCC GG- shown in SEQ ID NO.l 1,

-CAC CAC AGC CTA GAC ACC GG- shown in SEQ ID NO.12,

-CTC CCA GCA CTG ATC GTT TAG- shown in SEQ ID NO.13,

-CCG CTA CTC CGG AAA TTC- shown in SEQ ID NO.14,

-CTA CTC CGG AAA TTC TGC- shown in SEQ ID NO.15,

-GAC CAG CTA CGC GTC GTC G- shown in SEQ ID NO.16,

-TCT CAG ACC AGC TAG GCG- shown in SEQ ID NO.17,

-CGC CAC TAT CCC CGA AGG G- shown in SEQ ID NO.18,

-CCC TGA CTT AAA AGG CCG- shown in SEQ ID NO.19,

-AAG GGA TCG TTC GAC TTG- shown in SEQ ID NO.20,

-TGG GGA TAG ACC CTC TGT C- shown in SEQ ID NO.21,

-GCC ATG AAG ACG CCC GCT C- shown in SEQ ID N0.22,

-CCC GAA GCA GAA GGC ACG- shown in SEQ ID N0.23,

-CGC CGC TGA CGC CAT GAA- shown in SEQ ID N0.24,

-TGC CTC CTC TCG CGA GGT- shown in SEQ ID N0.25,

-CAC GGG CAG TTC CCT CAG- shown in SEQ ID NO.26, -CCC CCA GTT TCC CAA GGC- shown in SEQ ID N0.27,

or a functional variant thereof.

18. An assembly kit for monitoring the presence of agglomerate- and/or biofilm- forming microorganisms in a paper or board making process, characterized in that said kit comprises one or more oligonucleotide probes hybridizable with biofilm- forming microorganism(s), preferably with primary biofilm-forming microorganism(s), occurring in paper and board industry and labelled with a detectable marker.

19. The assembly kit according to claim 18, characterized in that the kit further comprises

a support for receiving the sample and for fixing the sample thereon,

reagents for fixing the sample,

hybridizing buffer solution containing the oligonucleotide probe(s) hybridizable with the target biofilm-forming microorganism(s), prefereably with primary biofilm-forming microorganism(s), occurring in paper and board industry and labelled with a detectable marker,

washing buffer solution, and optionally

unlabelled non-specific oligonucleotide(s) for blocking any unspecific binding sites

20. The assembly kit according to claim 18 or 19, wherein the kit comprises one or more probes as defined in claim 17.

21. The assembly kit according to any of claims 18-20, wherein the support for receiving the sample is a sampler support, e.g. a test plate, to be placed in the process for enabling the biofilm-forms to form a biofilm on the surface thereof in situ in the process, to be used as a support for the fixation, i.e. permeabilization, of the microorganisms present in the biofilm formed on the surface thereof and for the hybridization of the oligonucleotide probe(s) with the target microorganism(s) present in the biofilm formed on the surface thereof.

22. The assembly kit according to any of claims 18-21, wherein the kit further comprises miniature hybridization chamber(s) to be attached, e.g. glued, on the support.