Kağıt endüstrisi atıksularında anaerobik mikrobiyal çeşitliliğin belirlenmesi

Orhan İNCE, Şükriye ÇELİKKOL, Bahar Kasapgil İNCE, Zeynep ÇETECİOĞLU, Mustafa KOLUKIRIK

Özet

Bu çalışmada, kâğıt endüstrisi atıksularını arıtan gerçek ölçekli bir anaerobik kontak reaktörün 3 farklı yüksekliğinden 2 farklı zamanda alınan çamur numunelerinin mikrobiyal komünite yapıları Denatüran Gradyan Jel Elektroforez (DGGE) yöntemi kullanılarak karşılaştırılmıştır. 2 aylık izleme dönemi içinde sistem 2 hafta süreyle bakıma alınmıştır. Kontak reaktörün 1.6-1.8 kg KOİ/m3.gün organik yükleme hızında, KOİ giderim verimi % 47-55, metan üretim verimi 0.18-0.20 m3CH4/kgKOİgiderilen aralığında değişmiştir. DGGE analizleri sonucu, arkeyal popülasyona ait 31, bakteriyel popülasyona ait 57 farklı tür tespit edilmiştir. Arkeyal popülasyona ait 3 farklı tür Ağustos 2005’te tespit edilememiş, buna karşın 6 yeni tür gözlenmiştir. Bakteriyel popülasyonda ise Temmuz 2005 numunesine ait 10 farklı tür Ağustos 2005 numunesinde tespit edilemezken Ağustos 2005’te 10 yeni türün varlığı gözlenmiştir. Bu çalışmada incelenen reaktöre ait asetoklastik metan üretim kapasitesi önceki bir çalışmada Spesifik Metan Aktivite (SMA) test düzeneği kullanılarak ölçülmüş ve potansiyel metan üretiminin yaklaşık % 45 azaldığı tespit edilmiştir. Sistemde bulunan metanojenlerin ve Sülfat İndirgeyici Bakterilerin (SRB) tür ve sayıları Floresanlı Yerinde Hibritleme (FISH) yöntemi ile belirlenmiştir. SMA testi ve FISH tekniği ile tespit edilen mikrobiyal komünite değişimi DGGE yöntemi ile de doğrulanmıştır. DGGE yöntemi, iki farklı zamanda alınan numunelere ait komünite değişimini açıkça yansıtmakla birlikte sayısal değerlendirmede yetersiz kalmaktadır. Bu nedenle, anaerobik reaktörlerin mikrobiyal komünite yapılarının gerek DGGE gibi detaylı kalitatif sonuç veren gerekse FISH gibi mikroskobik sayıma dayalı, kültürden bağımsız yöntemlerle çalışılması gerektiği sonucuna varılmıştır.

Abstract

The use of anaerobic technologies in the fields of wastewater treatment, sludge stabilization, bioremediation and management of hazardous and solid wastes has grown in importance during the last few decades. Although the general processes occurring in anaerobic biological wastewater treatment plants, such as hydrolysis, fermentation, acetogenesis, methanogenesis and sulfidogenesis are well understood, the microbial community responsible for these conversions is often considered as a black box. Physical and chemical parameters only give rough estimations about the operational conditions of the system. Therefore, understanding the biodiversity and the dominant species of the microbial community is of great importance in studying contaminant degradation pathways, optimizing treatment processes, and improving removal efficiencies of engineer- designed systems. The culture dependent methods used for the investigation of the biomass are not sufficient for the identification of the complex microbial diversity in wastewater treatment systems. The use of the culture- independent methods in microbial ecology allowed the determination of the complex microbial populations and community contents more representatively. Denaturing Gradient Gel Electrophoresis (DGGE) is a Polymerase Chain Reaction (PCR) dependent method used for the electrophoretic separation of the 16S rDNA genes due to the difference in the nucleotide sequences. The separation is observed as an individual band on the DGGE gel. Every DGGE band represents a single species and the DGGE pattern gives the fingerprint of that community. Since DGGE technique allows the analysis of many samples simultaneously and gives rapid results, the use of DGGE is getting extensive in the investigation of bioreactors and natural ecosystems which inhabit rich microbial diversity. In the context of this study, a full-scale anaerobic contact reactor treating pulp and paper mills effluents was investigated. Samples were taken from 3 different levels at 2 different times. There was a 2- week off-period of the reactor between sampling times. Denaturing Gradient Gel Electrophoresis (DGGE) was used for the fingerprinting of the microbial community in the sludge samples. Performance of the reactor in terms of COD removal efficiency and methane yield varied between 47% and 55% and 0.18 and 0.20 m3CH4/kgCODremoved at Organic Loading Rates (OLRs) in a range of 1.6-1.8 kg COD/m3day, respectively. DGGE analysis revealed that 31 species from archaeal population and 57 species from bacterial population were present in the anaerobic reactor. 3 species from the archaeal population were not detected in August 2005 whereas 6 species were newly observed. In bacterial population, 10 species belonging to July 2005 samples were not detected where 10 other species were found in August 2005. Acetoclastic methanogenic activity of the reactor had previously been investigated by specific Methanogenic Activity (SMA) test. A decrease of 45% in the potential methane production was observed during the monitoring period of 2 months. The quantities and species of methanogens and Sulfate Reducing Bacteria (SRB) in the reactor were determined by Fluorescent In Situ Hybridization (FISH). Parallel to SMA results, the quantities of SRB and methanogens were decreased in August 2005. The shift in the microbial community observed by SMA test and FISH quantifications were supported by DGGE analysis. During the monitoring of 2 months, 2 weeks shut-down of the anaerobic reactor might have caused activity loss and microbial community change. DGGE allows the comparison of microbial communities taken from the anaerobic reactor at two different sampling times and FISH informs quantities of present microbial species in the reactor. However, DGGE does not give numerical information but clearly depicts the community shift. Therefore, it is concluded that, the microbial community structures of anaerobic reactors should be determined by culture independent methods of both qualitative and quantitative techniques such as DGGE and FISH respectively.