Analysis of the microbial community structure and function in a paper mill water system

Abstract

English: Microbiological studies of wastewater treatment systems generally rely on methods that are dependent on the culturability of the microorganisms being investigated. However, it has recently been estimated that less than 1 % of all microorganisms are culturable on synthetic media and under artificial conditions. The numbers of microorganisms are, therefore, largely underestimated. Due to the limitation of the conventional microbiological methods, numerous alternative assays have been proposed. These assays include the analysis of the functional and structural diversities of microbial communities in situ. A study was, therefore, undertaken at the Sappi Cape Kraft paper mill, Milnerton, Cape Town, South Africa, to evaluate the applicability of these alternative monitoring techniques in industrial systems. Microbial contamination at the plant are substantially enhanced due to the reuse of water and the use of recycled fibre for the production of fluting and linerboard. The influence of the production of various paper grades, biocide application and monthly shuts on the functional diversity of the microbial communities within the water system was determined. Results obtained during this study indicated that different microbial communities developed during the production of the different paper grades. A difference in the functional diversity of the planktonic and the sessile communities was evident after a change in the production grade. The shift in the functional diversity of the microbial community present in the planktonic phase was evident almost immediately upon a change in the board grade. Although a shift in the functional diversity of the microbial community within the sessile phase was also evident, the response was significantly delayed. The effect of different biocide dosages was also monitored in the planktonic and the sessile phases. No clear trends concerning the period of time after a shutdown could be observed in the planktonic phase, although a change could be observed in the sessile phase. It was concluded that the analysis of the functional diversity was sensitive enough to detect shifts in the community and to differentiate between microbial communities within the same system. The shifts could be ascribed to changes in the operational parameters in the water system. The influence of the process parameters on the structural diversity of the microbial community was also determined using signature lipid biomarkers. The ratio of diglyceride fatty acids to phospholipid fatty acids provided an estimate of the nonviable to viable microorganisms in the water system. A high biomass mortality was generally observed throughout the one year period of evaluation. The high ratios of trans to cis C18:1 obtained during this study were also indicative of stressed microbial communities. Based on the presence of signature lipid biomarkers, it was concluded that a large diversity of microorganisms was present in both the sessile and planktonic phases. The same trends in the number of cultured cells and the counts obtained with PLFA analysis were observed in all samples. Differences in the abundance and groups of specific fatty acids were detected due to the production of different board grades. These results confirmed the shift in the microbial community, as was detected on the basis of substrate utilisation profiles. Based on the results obtained during this study, it is evident that the application of signature lipid biomarkers provided substantially more information on the microbial communities than conventional culturing. Due to these positive results, the new techniques were applied to characterise the functional and structural diversities of the microbial communities during two microbial audits at the Sappi Cape Kraft paper mill. The first audit was performed to assess the efficiency of the current microbiological control programme at the mill and to make recommendations for the improvement of the microbial control. The second audit was performed to evaluate the effect of subsequent changes made to the microbial control programme. Due to the indication that resistance to the current biocides had developed, alternative biocides were recommended for future use in the system. A large diversity of microorganisms was detected in the various unit operations using phospholipid fatty analysis. Cluster analysis of the functional diversity of the various microbial communities indicated that groups of unit operations could be closed using separate biocide dosage programmes. It may thus be concluded that the application of these alternative techniques provided substantial information concerning the microbial function and structure in situ. This information assisted in the implementation of an alternative microbiological control programme at the paper mill, which resulted in a significant reduction in the microbiological associated problems.

Afrikaans: Mikrobiologiese studies en evaluasie van programme om watersisteme te behandel maak oor die algemeen staat op metodes wat afhanklik is van die kweekbaarheid van mikroorganismes. Daar is egter onlangs beraam dat minder as 1 % van alle mikroorganismes op sintetiese media en onder kunsmatige toestande gekweek kan word. Die getal mikroorganismes word daarom tot 'n groot mate onderskat. As gevolg van die beperkings waaraan konvensionele mikrobiologiese metodes onderhewig is, is verskeie alternatiewe metodes dus voorgestel. Hierdie metodes sluit die ontleding van die funksionele- en strukturele-diversiteit van mikrobiese gemeenskappe in hulle natuurlike toestand in. 'n Studie om die toepaslikheid van hierdie alternatiewe moniteringstegnieke in indurstriële stelsels te evalueer, is dus by die Sappi Cape Kraft papiermeule te Milnerton, Kaapstad onderneem. Mikrobiese ) kontaminasie van die aanleg word aansienlik vererger as gevolg van die hergebruik van water en die gebruik van herwinde vesels in die vervaardiging van riffel- en voering-karton. Die invloed van verskillende karton produkte, die aanwending van biosiedes en maandelikse sluiting vir instandhouding op die funksionele-diversiteit van die mikrobiese gemeenskappe in die waterstelsel is bepaal. Dit het geblyk dat daar 'n verskil in die funksionele-diversiteit van die planktoniese en sessiele gemeenskappe ingetree het na 'n verandering in die karton graad. Hoewel dit geblyk het dat daar ook 'n verandering in die funksionele-diversiteit van die mikrobiese gemeenskap in die sessiele fase voorgekom het, was die reaksie aansienlik vertraag. Die uitwerking van verskillende biosiedes op die planktoniese en sessiele fases is ook gemonitor. Geen duidelike tendense betreffende die tydsverloop na 'n sluiting kon in die planktoniese fase onderskei word nie, hoewel "n verandering in die sessile fase waargeneem is. Die gevolgtrekking was dat die ontleding van die funksionele diversiteit sensitief genoeg was om veranderings in die mikrobiese gemeenskap vas te stel en om te onderskei tussen verskillende gemeenskappe in dieselfde stelsel. Die veranderings kan toegeskryf word aan veranderings In die operasionele parameters In die waterstelsel. Die invloed van die prosesparameters op die strukturele diversiteit van die mikrobiese gemeenskap is ook bepaal deur gebruik te maak van indikator-lipiede as biomerkers. Die verhouding van digliseried vetsure tot fosfolipied vetsure het dit moontlik gemaak om 'n raming te maak van die lewensvatbaarheid van die mikroorganimses in die waterstelsel. 'n Hoë tempo van afsterwing is waargeneem oor 'n tydperk van een jaar. Die hoë verhouding van trans tot cis CIS:1 wat gedurende hierdie studie waargeneem is, was ook 'n aanduiding van mikrobiese gemeenskappe wat onder omgewings druk was. Die aanwesigheid van spesifieke indikator-lipiede het daarop gedui dat 'n groot diversiteit van mikroorganismes in beide die sessiele en planktoniese fases aanwesig was. Dieselfde tendense in die getal kweekbare selle is verkry as met die getalle wat met behulp van fosfolipied vetsuur analises bepaal is. Verskille wat in die hoeveelhede van spesifieke groepe vetsure vasgestel is, was die gevolg van die vervaardiging van verskillende tipes karton. Hierdie resultate het die verandering in die mikrobiese gemeenskap bevestig, wat bepaal is deur die profiele van substraatbenutting. Uit die resultate wat gedurende die studie verkry is, blyk dit dat die gebruik van indikator-lipiede aansienlik meer inligting verskaf oor die mikrobiese gemeenskappe as wat met konvensionele kweking moontlik is. Op grond van die positiewe resultate wat verkry is, is hierdie alternatiewe tegnieke gebruik om die funksionele- en strukturele-diversiteit van die mikrobiese gemeenskappe gedurende twee mikrobiese oudits by die Sappi Cape Kraft papiermeule te karakteriseer. Die eerste oudit is uitgevoer om die doeltreffendheid van die huidige mikrobiologiese beheerprogram by die meule te evalueer en aanbevelings te maak orn die beheerprogram te verbeter. Die tweede oudit uitgevoer is om die uitwerking van die veranderinge aan die mikrobiese beheerprogram te evalueer. As gevolg van die aanduidings dat weerstand teen die biosiedes wat aangewend is ontwikkel het, is aanbeveel dat alternatiewe biosiedes in die toekoms toegedien word. 'n Wye verskeidenheid van mikroorganismes is met fosfolipied vetsuuranalises in die verskillende eenheids prosesse aangetref. Trosanalise van die funksionele-diversiteit van die verskillende mikrobiese gemeenskappe het aangedui dat verskillende prosesse saam gegroepeer kon word om met soortgelyke biosiedprogramme behandel te word. Die gevolgtrekking was dat die aanwending van hierdie alternatiewe tegnieke uiters belangrike inligting betreffende die mikrobiese funksie en stuktuur in situ verskaf het. Hierdie inligting het meegehelp om 'n alternatiewe mikrobiologiese beheerprogam by die meule te implementeer, wat aansienlike vermindering in probleme teweeggebring het wat verband hou met mikrobiese kontaminasie.