Anoxygenic Phototrophic Bacterial Diversity Within Wastewater Stabilization Plant During ‘Red Water’ Phenomenon
Total Page：16
File Type：pdf, Size：1020Kb
Int. J. Environ. Sci. Technol. (2013) 10:837–846 DOI 10.1007/s13762-012-0163-2 ORIGINAL PAPER Anoxygenic phototrophic bacterial diversity within wastewater stabilization plant during ‘red water’ phenomenon A. Belila • I. Fazaa • A. Hassen • A. Ghrabi Received: 22 November 2011 / Revised: 2 October 2012 / Accepted: 21 October 2012 / Published online: 13 February 2013 Ó Islamic Azad University (IAU) 2013 Abstract The molecular diversity of the purple photo- Introduction synthetic bacteria was assessed during temporal pigmen- tation changes in four interconnected wastewater Wastewater stabilization ponds (WSPs) are an extremely stabilization ponds treating domestic wastewater by dena- effective, natural form of wastewater treatment. They turant gel gradient electrophoresis method applying pufM combine simplicity, robustness, low cost, and a very high gene. Results revealed high phylogenetic diversity of the degree of disinfection. WSPs are usually designed as one or purple phototrophic anoxygenic bacteria community char- more series of anaerobic, facultative, and maturation acterized by the presence of the purple non-sulfur, purple ponds. Their low operation and maintenance costs have sulfur, and purple aerobic photosynthetic anoxygenic bac- made them a popular choice for wastewater treatment, teria. This phototrophic bacterial assemblage was domi- particularly in developing countries since there is little nated by the purple non-sulfur bacteria group (59.3 %) need for specialized skills to operate the system. One of the with six different genera followed by the purple sulfur major problems which can cause malfunction of the WSP community (27.8 %) with four genera and finally 12.9 % is the massive growth of purple color producing bacteria of the pufM gene sequences were assigned throughout the (Belila et al. 2009; Sirianuntapiboon and Srikul 2006). aerobic anoxygenic phototrophic bacterial group. The When ponds were overloaded under anoxic and/or facul- purple phototrophic bacterial community was characterized tative conditions, the wastewater color appears purple in by the presence of salt-dependant bacterial species color due to the toxic effects of mainly sulfide on the algae belonging to the genus Marichromatium, Thiorhodococcus, and the consequent predominance of purple anoxygenic Erythrobacter, and Roseobacter. The wastewater treatment photosynthetic bacteria (Veenstra et al. 1995; Villanueva plant performances were unsatisfactory, and the biological et al. 1994). As a result of this massive growth, chemical and chemical parameters suggested that the purple photo- and physical wastewater effluent qualities such as color, synthetic bloom was correlated with the eutrophic state. smell, and suspended solid concentration can be seriously affected (Nair 1992). Keywords Functional diversity Á Eutrophic state Á The phototrophic purple bacteria are extremely hetero- Domestic wastewater Á Denaturant gel gradient geneous on basis of morphological, physiological, and electrophoresis Á pufM gene molecular data. Among the six phyla belonging to the photosynthetic prokaryotes, the purple phototrophic anoxygenic bacteria are the most metabolically diverse group. Currently, three groups of anoxygenic phototrophic bacteria are established: the purple sulfur bacteria (PSB), the purple non-sulfur bacteria (PNSB), and the aerobic A. Belila (&) Á I. Fazaa Á A. Hassen Á A. Ghrabi anoxygenic phototrophic purple bacteria. Due to the wide Water Treatment and Reuse Laboratory, Water Researches distribution of this physiological bacteria group through and Technologies Center, Bordj-Cedria, BP. 273, 8020 Soliman, Tunisia several class of the Proteobacteria and due to their close e-mail: [email protected]; [email protected] phylogenetic relationship to non-phototrophic bacteria, the 123 838 Int. J. Environ. Sci. Technol. (2013) 10:837–846 use of general bacterial 16S rRNA gene primers often underestimates the diversity of phototrophic bacteria espe- cially those present in rare numbers. The cultivation- dependent approaches also are critical and may lead to significant gaps in microbial diversity studies (Donachie et al. 2007). Different molecular methods based on the pufM gene encoding the M subunit of the photosynthetic reaction center have been used to explore the phototrophic bacterial diversity (Jiao et al. 2007) such as gradient gel electropho- resis (Lehours et al. 2010), terminal restriction fragment length polymorphism (Ranchou-Peyruse et al. 2006), and shotgun sequencing (Yutin et al. 2007). Although these DNA-based approaches have given insight into the diversity of anoxygenic phototrophic bacteria (APB), analysis of relevant functional communities is still a challenge. So far, most studies on WSP systems have concentrated on the fate and removal of pathogenic microorganisms, Fig. 1 Schematic representation of the wastewater stabilization such as fecal enterococci (Reinoso and Be´cares 2008) and plant. AP anaerobic pond, FP facultative pond, MP maturation pond helminthes eggs (Tyagi et al. 2008). However, this study presents for the first time detailed taxonomic and phylo- genetic information concerning the purple phototrophic Table 1 Geometric characteristics of the wastewater stabilization ponds microbial community composition during this bacterial photosynthetic bloom. For this purpose, a PCR-DGGE Anaerobic Facultative Maturation Maturation pond pond pond 1 pond 2 based approach targeting specific structural gene pufM and photopigments analysis were realized within four waste- Surface (m2) 30 100 122 129 water stabilization ponds situated in the North East of Depth (m) 3.5 2.4 1.34 1.22 Tunisia exhibiting periodically red water phenomenon in Volume (m3) 96 180 164 147 April 2009. The ecophysiological factors enhancing this Water 3.3 2 1.15 1 bacterial event were also discussed. depth (m) Materials and methods facultative (SF1–SF4) ponds with Plexiglas core tubes (5 cm Wastewater stabilization plant description diameter, 25 cm length). The maturation pond sediment layer was very thin and could not be sampled. Four water This study was carried out in four WSPs located in Mutu- samples were also collected (WA,WF,WM1 and WM2) and elleville urban City (36°490 N, 10°100 E) in the North East were filtered using 0.2 lm pore size filters (Millipore). For of Tunisia. The wastewater stabilization system consists of molecular analysis, all samples were frozen at -50 °C. four inter-connected ponds: the anaerobic pond followed by the facultative and two maturation ponds (Fig. 1). Size and Physical and chemical parameters depth of these ponds are summarized in Table 1. Effluents are essentially of domestic origin and result essentially from In situ measurements of the temperature and pH were black waters (waters used for toilets) as well as grey waters performed using WTW Handheld Meters 340i model (domestic sewage). Preliminary and primary treated (WTW, Weilheim, Germany). Dissolved oxygen (DO) wastewater fills up the first pond (anaerobic pond: An) and concentration was carried using a Multiline F/set P4 uni- then enters the secondary facultative (F) pond through an versal meter (WTW). Five-day biochemical oxygen outflow, and finally to the maturation (M) pond. demand (BOD5), chemical oxygen demand (COD), total suspended solids (TSS), and sulfate and sulfide concen- Sampling procedure trations were carried out according to the analytical methods described in standard methods for the examination A total of 12 samples were collected from the wastewater of water and wastewater (APHA 1995). The concentration stabilization ponds in April 2009. Four sediment samples of the chlorophyll a was estimated by the methanol were extracted from the anaerobic (SA1–SA4) and the extraction method (Pearson 1986). 123 Int. J. Environ. Sci. Technol. (2013) 10:837–846 839 Enrichment of the phototrophic purple bacteria denaturant (100 % denaturant was 7 M urea and 40 % and photopigments analysis (v/v) formamide) at 130 V for 8 h at 60 °C (Karr et al. 2003). DGGE gel was stained with Sybr GreenI (Sigma- Aliquot volumes (1 ml) of wastewater collected from each Aldrich Corporation, St. Louis, MO, USA) and was visu- wastewater stabilization pond were used to inoculate alized on a UV transilluminator. Individual bands were enrichment cultures for anoxygenic phototrophic bacteria. excised, re-suspended in 20 ll of Milli-Q water, and stored Two enrichment media were prepared according to the overnight at 4 °C. A volume of 3–5 ll of the supernatant method of Pfennig and Tru¨per (1992) and Weaver et al. was used for re-amplification using the original PCR con- (1975) to enrich purple sulfur and non-sulfur bacteria, ditions and the same primer pair without a GC clamp and respectively. The enrichment cultures were incubated at then photographed using a GBox iChemi 2D gel image 30 °C with light intensity of 500 lux from a tungsten lamp analyzer using Genesnap software 7.03 (Syngene, Synop- (16 h light and 8 h dark). In-vivo absorption spectra of living tics, Ltd, Cambridge, UK). cells were determined using a Unicam Helios, a scanning spectrophotometer of a suspension of the cell pellets pre- Sequencing of DGGE bands pared in a sucrose solution (Pfennig and Tru¨per 1992). Prior to the sequencing reactions, primers were enzymati- Nucleic acid extraction and PCR amplification cally removed from the reamplified DGGE bands using the ExoSAP-ITTM kit (Amersham Bioscience, Roosendaal, Total genomic DNA from sediment and water samples was The Netherlands) following the descriptions of the manu-