REV.CHIM.(Bucharest)♦70♦No. 1 ♦2019 http://www.revistadechimie.ro 293 Microbial Diversity of Aerobic Granular Sludge under Different Operational Conditions COSTEL BUMBAC, ELENA ELISABETA MANEA*, MIHAI NITA-LAZAR, OLGA TIRON National Research and Development Institute for Industrial Ecology -ECOIND, 71-73 Drumul Podul Dambovitei Str., 060652, Bucharest, Romania This paper depicts the research conducted at lab scale on aerobic granular sludge sequential biological reactors for wastewater treatment in order to assess the influence of operational parameters on the treatment performances and microbial diversity of the granules. The proposed objectives were reached by testing in similar reactor conditions different organic nitrogen and phosphorus loading rates. The study focused both on treatment performances as organic, nitrogen and phosphorus removal based on chemical analysis of main quality parameters and on diversity of microbial population in granules based on real-time polymerase chain reaction by targeting mainly the specific species or functional genes with high influence on process performance in terms of nitrification, denitrification and phosphorus removal. Keywords : aerobic granular sludge, denitrification, nitrification, real-time PCR. Aerobic granular sludge has recently gained the reputation of an all-in-one biological system encompassing different process conditions (aerobic, anoxic, anaerobic) thus allowing the microorganisms within its structure to simultaneously be capable of organic matter, nitrification, denitrification and phosphorus removal in one reactor [1- 3] while other processes need several reactors [4,5]. Nitrification-denitrification and shortcut processes variations of these are mainly responsible for nitrogen compounds removal in wastewater treatment systems. Nitrification is the biological oxidation of ammonia or ammonium to nitrite followed by the oxidation of the nitrite to nitrate [6]. The oxidation of ammonia into nitrite is performed by two groups of organisms, ammonia- oxidizing bacteria (AOB, e.g. by Nitrosomonas sp. or Nitrosococcus sp.) [7] and ammonia-oxidizing archaea (AOA, e.g. by Nitrosopumilus sp. and Nitrososphaera sp.) [8]. Ammonia oxidizing bacteria use two enzymes, ammonia monooxygenase (a multisubunit enzyme encoded by the amo C, amo A, and amo B genes [9]) and hydroxylamine oxidoreductase, to oxidize ammonia to nitrite via hydroxylamine as an obligate intermediate [10]. Further oxidation of nitrite to nitrate is performed by nitrite oxidoreductase (NXR) considered to be the best candidate as specific functional marker for nitrite oxidizing bacteria (NOB) [11]. This membrane-associated enzyme occurs in two phylogenetically distinct forms, one cytoplasmic type found in the NOB Nitrobacter, Nitrococcus and Nitrolancetus (Sorokin et al., 2012), and one periplasmic type found in Nitrospira and Nitrospina [11,12]. Denitrification is the biological process by which nitrate is reduced to nitric oxide (NO), nitrous oxide, and, finally, dinitrogen, using a series of nitrogen oxide reductases NirK (or NirS), NorB, and Nos Z, respectively [13]. The reduction of nitrite by nitrite reductase ( Nir) is the key step in the denitrification pathway [14]. Two types of structurally different but functionally equivalent nitrite reductase that are translated from the nirS and nirK genes are considered to be the predominant enzymes for catalyzing nitrite reduction [13]. Biological phosphorus removal in wastewater treatment plants is achieved by phosphate accumulating bacteria (PAOs) - a wide range of bacteria able to accumulate polyphosphates as a phosphate reserve [15]. Either we talk about conventional or granular sludge, recent research efforts have stressed that optimization of the microbial community structure and functioning should be a major objective in the design and operation of a treatment system [16-18]. Most efforts are being made for process optimization through monitoring of chemical (e.g. NH 4 + ; NO 2 - , dissolved oxygen, p H) and physical (e.g. flow rate, temperature) parameters in order to enhance the process safety and the biological reactions. However, a consistent long-term performance can only be ensured when the microbial community within the sludge functions optimally. For this purpose, questions about community structure, activity and the population kinetics have to be answered by means of molecular monitoring tools, which allow to identify and quantify the microorganisms present in the sludge (conventional or granular) of wastewater treatment plants [6,17]. In this study, we monitored the microbial composition of the aerobic granular sludge using real-time polymerase chain reaction (rt-PCR) by targeting mainly the specific species or functional genes with high influence on process performance in terms of nitrification, denitrification and phosphorus removal. Experimental part The experiments for wastewater treatment were performed in a lab scale aerobic granular sludge sequential biological reactor with a total working volume of 6 L. The bioreactor was used to evaluate the impact of different organic loading rates on treatment performances and on the microbial diversity of aerobic granular sludge. The experimental setup consisted of: influent vessel (30 L), feeding pump (Heidolph, PUMPDRIVE 5001 peristaltic pump), column type bioreactor, effluent vessel (30 L). The cyclic operation of the bioreactor was ensured by computer-based control system which controlled the feeding pumps, air inlet and effluent outlet electro valves. Total HRT of the bioreactor 8 hours, with the following operational time sequence: anaerobic feeding (10 min.), aerobic reaction (7h 35 min.), settling (5min.) and effluent * email: elena.manea@incdecoind.ro