Effect of sludge age on the diversity of nitrification bacteria and reactor stability

Çamur bekletme süresinin nitrifikasyon sistemlerindeki bakteriyel çeşitliliği ve sistem stabilitesine etkisi

Abstract

Variations in the relative abundance of microbial species are proved responsible of problems in biological treatment reactors. Quantitative ecology of wastewater treatment processes is the way of understanding these variations in community composition. In this point of view, many studies have been performed not only to understand the microbial structure of a wastewater treatment plants, but also to link microbial community dynamics to process stability. There are studies which investigated the instability caused by environmental conditions using lab- and full-scale treatment reactors. However, it is still unclear that functional stability implies a persistent community. Purkhold et al. (2000) found, for example, that few of the nitrifying bacteria classically studied in the laboratory were present in fullscale nitrification systems. and functionally stable ecosystem. Saikaly et al. (2005) studied the stability of the treatment performance of laboratory scale sequencing batch reactors operated under different sludge ages. Results of the work by Fernandez et al. (1999) revealed that an extremely dynamic community sustains a more reliable treatment performance in terms of stable efficiency. However, similar studies revealed differences in the community structure of any changes in AOB community structure. However, it is not clear whether persistent community composition secures to get stable treatment performance. This is especially important for the microorganisms which have lower growth rate (e.g. nitrifiers) as they would be less abundant compared to others (e.g. heterotrophs). Loss or alteration of key functional groups might cause unstable treatment performance. Therefore, nitrification process is selected as a main investigation subject in the present study. Samples were collected for chemical analysis and also for molecular analysis. FISH and DGGE were applied to reveal total AOB numbers and any changes in community composition. To achieve this, two sets of replicate reactors were operated under identical conditions under two different sludge ages (3 and 10 days) with the same external conditions. During the whole investigation period, reactor 1 and 2 (3 days sludge age) achieved more than 88% COD removal whilst reactor 3 and 4 (10 days sludge age) achieved more than 96% COD removal. Results also showed that replicate reactors with high sludge age performed more stable nitrification performance than the reactors with low sludge age. Nitrification performance was assessed with ammonium, nitrite and nitrate concentrations. In reactor 1 and 2, nitrate concentrations were in a range of 0 and 28.48 mg/L; whilst it was up to 40 mg/L in reactor 3 and 4. Nitrite concentration was maximum 10 mg/L in reactor 1 and 2; whereas no higher than 2.5 mg/L in reactor 3 and 4 during the first 30 days of experiment. FISH quantification revealed no correlation in total AOB numbers within replicate reactors. DGGE analysis also revealed decreasing similarities between replicate reactors over time. This was more obvious in reactors with higher sludge. On the day 32, the similarity values were 70.6% and 26.7% between reactor 1-2 and reactor 3-4, respectively. Beside statistical analysis, predominant bands were excised from DGGE gels and sequenced. Results showed that they match to the following sequences: Nitrosomonas oligotropha (97.6 % similarity; accession number: AF272422); Nitrosomonas sp. IS79A3 (97.4% similarity,accession number: AJ621026) and uncultered beta-proteobacteria (higher than 97.7% similarity; accession numbers: AY062126, DQ413103, DQ376558, AY064177). Sequencing results were in aggrement with the literature which states that Nitrosomonas species are dominant in wastewater treatment reactors. This study is of particular importance as there is still a gap in knowledge on performance stability and community composition in wastewater treatment reactors. Since, replicate reactors were run under identical conditions, our findings could be helpful to solve the link between performance and microbial diversity. Furthermore, we hope that our findings would be in use for designing treatment reactors with new strategy.

Özet

Bir arıtma tesisinin başarısı büyük ölçüde arıtma işini yapacak mikrobiyal komünitenin oluşumuna bağlıdır. Fonksiyonel öneme sahip populasyonların kaybolması veya aktivitesini yitirmesi arıtma sisteminde verim kaybına yol açar. Kısaca, bir sistemin kararlı halde çalışması sahip olduğu mikroorganizma türlerinin sistemde kararlı halde tutulabilmesine bağlıdır.Yeterli miktarda ve ceşitlilikte mikroorganizmanın sistemde tutulabilmesi, optimum çamur yaşının belirlenebilmesi ile mümkündür. Ancak, son yıllarda yapılan çalışmalarla, çevre veya işletme koşullarında bir farklılık olmadığı durumlarda dahi arıtma sistemlerinin bir süre sonra stabilitesini kaybettiği gösterilmiştir; ancak stabil (kararlı) arıtma verimi elde etmek için, kararlı bir komünite yapısına sahip olmak gerektiği hala kesinlik kazanmamıştır. Bu durum, düşük büyüme hızına sahip nitrifikasyon bakterileri için daha fazla önem arz etmektedir. Bu çalışmada nitrifikasyon prosesi model olarak seçilmiş ve bu sistemlerin kararlı yapısı üzerine araştırma yapılmıştır. Büyüme hızı ile doğrudan ilişkilendirilmesi nedeniyle çamur yaşı bifürkasyon parametresi olarak seçilmiş ve laboratuar ölçekli atıksu arıtma reaktörleri farklı iki çamur yaşında işletilmiştir. Kimyasal analizler yanında moleküler analizler kullanılarak reaktörlerdeki nitrifikas-yon bakterilerinin sayısı ve yapısındaki değişimler incelenmiştir. Elde edilen sonuçlar, replika reaktörlerde toplam Amonyağı Oksitleyici Bakteri (AOB) sayısı açısından anlamlı bir ilişki olmadığını göstermiştir. Bunun yanında; bütün 3 gün çamur yaşına sahip reaktörlerde, 10 gün çamur yaşına sahip reaktörlere göre AOB çeşitliliğinin daha fazla olduğu, ancak zamana bağlı olarak çeşitliliğin azaldığı görülmüştür. Çeşitlilikteki bu azalma, yüksek çamur yaşında işletilen reaktörlerde daha fazla gözlenmiştir. Dizi analizi sonucu baskın olan türlerin Nitrosomonas türleri ve henüz kültüre alınmamış beta-proteobakteri olduğu bulunmuştur.