applied sciences Article Sequencing Batch Reactor and Bacterial Community in Aerobic Granular Sludge for Wastewater Treatment of Noodle-Manufacturing Sector Tang Thi Chinh 1,3,*, Phung Duc Hieu 1, Bui Van Cuong 1, Nguyen Nhat Linh 2, Nguyen Ngoc Lan 2,3, Nguyen Sy Nguyen 1, Nguyen Quang Hung 1,3 and Le Thi Thu Hien 2,3,* ID 1 Institute of Environmental Technology (IET), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 100000, Vietnam; [email protected] (P.D.H.); [email protected] (B.V.C.); [email protected] (N.S.N.); [email protected] (N.Q.H.) 2 Institute of Genome Research (IGR), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 100000, Vietnam; [email protected] (N.N.L.); [email protected] (N.N.L.) 3 Graduate University of Science and Technology (GUST), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, Hanoi 100000, Vietnam * Correspondence: [email protected] (T.T.C.); [email protected] (L.T.T.H.); Tel.: +84-904-187-106 (T.T.C.); +84-989-019-691 (L.T.T.H.) Received: 1 March 2018; Accepted: 26 March 2018; Published: 27 March 2018 Abstract: The sequencing batch reactor (SBR) has been increasingly applied in the control of high organic wastewater. In this study, SBR with aerobic granular sludge was used for wastewater treatment in a noodle-manufacturing village in Vietnam. The results showed that after two months of operation, the chemical oxygen demand, total nitrogen and total phosphorous removal efficiency of aerobic granular SBR reached 92%, 83% and 75%, respectively. Bacterial diversity and bacterial community in wastewater treatment were examined using Illumina Miseq sequencing to amplify the V3-V4 regions of the 16S rRNA gene. A high diversity of bacteria was observed in the activated sludge, with more than 400 bacterial genera and 700 species. The predominant genus was Lactococcus (21.35%) mainly containing Lactococcus chungangensis species. Predicted functional analysis showed a high representation of genes involved in membrane transport (12.217%), amino acid metabolism (10.067%), and carbohydrate metabolism (9.597%). Genes responsible for starch and sucrose metabolism accounted for 0.57% of the total reads and the composition of starch hydrolytic enzymes including α-amylase, starch phosphorylase, glucoamylase, pullulanase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, and 1,4-α-glucan branching enzyme. The presence of these enzymes in the SBR system may improve the removal of starch pollutants in wastewater. Keywords: aerobic granular sludge; sequencing batch reactor (SBR); bacterial community; noodle-manufacturing; wastewater treatment; predicted functional analysis 1. Introduction Among the wastewater treatment processes applied in recent days, sequencing batch reactor (SBR) has been considered as one of the most popular technologies. SBR is a type of activated sludge process that is comprised of aerobic or anaerobic digesters or mechanical biological treatment facilities in batches [1]. These systems generally require large surface areas for treatment and biomass separation units which enable the sludge to settle down, separating from the treated water. Therefore, the settling velocity of activated sludge plays an important role in reducing the remaining time in the settlement/decanting tank, and as a result, boosts the treatment process [2]. Conventional Appl. Sci. 2018, 8, 509; doi:10.3390/app8040509 www.mdpi.com/journal/applsci Appl. Sci. 2018, 8, 509 2 of 14 activated sludge systems are limited, due to the generally poor settling properties of the sludge [3]. To improve the ability to settle, new technology has been developed using aerobic granular sludge. Granules creating aerobic granular activated sludge are considered as aggregates of microorganisms, mainly bacteria, and extracellular polymeric substances [4]. Aerobic granular sludge also functions as a biofilm in suspension, which is composed of combining cells [5]. The application of granules for wastewater treatment has many advantages. They settle significantly faster than activated sludge flocs (high settling velocity). In addition, granular sludge provides high and stable rates of metabolism [5]. Activity of microbial community in granular sludge directly affects the effectiveness of the biological treatment of wastewater. Therefore, it is important to define the microbial composition as well as the interaction between species in an active treatment system [6]. Microbial diversity in a wastewater treatment plant is usually high with the appearance of bacteria, protozoa, metazoa and other microlife [7], but bacteria still do the major role in degrading organic pollutants in the treatment process. It is known that culture-dependent methods have limitations for studying natural microbial community composition, because only a small part of bacteria in environmental samples are culturable under laboratory conditions [8]. Molecular techniques such as polymerase chain reaction–denaturing gradient gel electrophoresis) (PCR–DGGE) [9–11], amplified ribosomal DNA restriction analyses (ARDRA) [12], and fluorescence in situ hybridization (FISH) have been widely applied to study the diversity of microorganisms in natural samples [13]. These techniques have significantly overcome the weakness of traditional methods, but only elucidate the major microbial groups in wastewater treatments. Recently, the metagenomic approach has emerged as one of the most effective and popular techniques for investigating microbial diversity and community in activated sludges [14–18]. The aims of this work are to develop a SBR system for wastewater treatment in a noodle-manufacturing village in Vietnam with high starch concentration, and to investigate the diversity of bacterial communities and their predictive functions presented in aerobic granular sludge, using MiSeq sequencing technique. The sludge used in this study is sampled from the SBR system conducted on a laboratory scale. 2. Materials and Methods 2.1. Wastewater Sample Collection for SBR System The wastewater from the noodle-manufacturing village of Phudo–Hanoi was collected and used as input material for the treatment process. The wastewater was characterized by parameters such as chemical oxygen demand (COD): 2500–3500 mg/L; biochemical oxygen demand (BOD5): 1700–2000 mg/L; total nitrogen (TN): 50–70 mg/L and total phosphorous (TP): 25–30 mg/L. The treatment process in the SBR system was supplemented with 50 mL of isolated inoculum Lactobacillus sp. (108 CFU/mL) to increase the density of useful microorganisms. 2.2. Operating Conditions of the SBR System The SBR system in this study had a total volume of 9.42 L and input COD ranging from 0.65 to 3 kg/m3·day). The pH of the wastewater was adjusted and remained at approximately 6.5–7.0, and the reactor was thoroughly stirred by air pumping dissolved oxygen (DO), with an O2 concentration of 5 mg/L, into the system. After the stability of granular sludge, the load of the system maintained a COD of around 4.5–7.5 kg/m3·day. The reactor was continuously operated for three hours for each batch. Initially, each round included 2 min for water and 166 min for air supply, then 10 min for settling and water pumping for another 2 min (Figure1). Appl. Sci. 2018, 8, 509 3 of 14 Appl. Sci. 2018, 8, x FOR PEER REVIEW 3 of 14 FigureFigure 1. DiagramDiagram of of the the sequencing batch reactor (SBR) system. Operation Operation process process starts starts with with pumpingpumping wastewater wastewater through through the the metering metering pump pump into into the the reactor. reactor. Gas Gas is is supplied supplied into into the the reactor reactor throughthrough air air pump pump and and the the outlet outlet water water is discharged via exhaust valve and collected in a container. 2.3.2.3. Collection Collection of of Activated Activated Sludge Sludge and and Outlet Outlet Water Water Analysis Analysis TheThe outlet outlet water water from from the the SBR SBR system system was was collected collected and and pooled pooled in ina container a container after after one one day day of operationof operation (equivalent (equivalent to 8 batches). to 8 batches). Then, the Then, outlet the water outlet samples water sampleswere collected were daily collected for analysing daily for parametersanalysing parameterssuch as suchCOD, as TN, COD, and TN, TP and concentrations TP concentrations according according to toStandard Standard Methods (APHA-AWWA-WPCF(APHA-AWWA-WPCF 1998)1998) [ 19[19].]. The The sludge sludge samples samples were were collected collected from from the SBR the system SBR system and filtered and filteredwith a membrane with a membrane of pore sizeof pore 45 µ sizem (MF 45 μm Millipore (MF Millipore® Membrane,® Membrane, MCE, 0.45 MCE,µm, 0.45 47 mm). μm, The47 mm). collected The collected sludge was also used to determine the sludge volume index at 30 min (SVI30), according the sludge was also used to determine the sludge volume index at 30 min (SVI30), according the method methodof Jenkins of etJenkins al. [20 et]. Dataal. [20]. were Data the were average the average values of values three of repetitions three repetitions for each for analysis. each analysis. ScanningScanning electron electron microscopy microscopy (SEM) (SEM) was was used used to to track track the the evolution evolution of of microbial microbial populations, populations, andand the the images images were were taken taken by by FE-SEM, FE-SEM, S4800, S4800,