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OPEN ACCESS Freely available online Applied Microbiology Open Access Research Article Profile of Nitrification and Denitrification Bacteria in the Different Layers of Filter Media in a Rhizofiltration System Mmasefako Lettie Mmammule Sikhosana1, Botha A2, Mpenyana-Monyatsi L1, Coetzee MAA1 1Department of Environmental, Water & Earth Sciences, Tshwane University of Technology, Pretoria, South Africa, 2Department of Microbiology, Stellenbosch University, Private Bag X1, Matieland, South Africa ABSTRACT The presence of nitrifying and denitrifying bacteria demonstrated the ability to remove nitrogen from the simulated urban runoff. The bacteria were isolated from the filter layers of the planted and unplanted sides of a constructed wetland. The system was operated with simulated urban runoff twice a week and was fed manually with a mixture of settled municipal wastewater. Culture-based methods and molecular techniques were applied to determine and identify the bacterial isolates sampled once in autumn from the different types of filter media used (three-layered filter). The 16S rDNA sequence analysis of the isolates recovered from the media revealed that the isolates were similar to the genus Pseudomonas, such as Pseudomonas yamanorum, Pseudomonas rhodesiae, and Pseudomonas extremaustralis. Isolates similar to Pseudomonas yamanorum were observed to be present in most filter layers of the planted side of the system and were also recovered in the second (middle) layer of the unplanted side of the system. Phylogenetic analysis confirmed the evolutionary relationship of isolates recovered in the layers of both the planted and unplanted sides of the systems to share similarities ranging between 95.6% and 100% with reference strains. The isolates were noted to possibly have nitrification abilities. Sequencing of genomic DNA also revealed that anaerobic denitrifying bacteria were present in the filter layers of both the planted and unplanted sides of the system. Evolutionary relationships of taxa also revealed that anaerobic denitrifying isolates recovered showed between 99.3% and 99.6% sequence similarity to reference strains. Conversion and removal of nitrogen is possible in the different layers of the filter media, namely sand and gravel, crushed stones and large stones, which function as a biological filter. Various heterotrophic nitrifying, aerobic denitrifying and facultative anaerobic bacteria carry out biological removal of nitrogen in the planted and unplanted sides of the system. Keywords: Nitrification; Denitrification; Heterotrophic nitrifiers; Aerobic denitrifiers INTRODUCTION used in achieving nitrogen removal in wastewater [3-5]. During the nitrification process, ammonium is oxidised to nitrite and then to Treatment of surface water, groundwater, industrial water, nitrate in a two-way step process by nitrifying bacteria in aerobic agricultural runoff, stormwater, acid mine drainage, urban runoff zones, while denitrification converts nitrate to nitrogen gas by and residential effluents has been achieved by the rhizofiltration denitrifying bacteria in anoxic and anaerobic zones [4-6]. process. This process involves the use of filter media and plants. Contaminants are removed when trapped into plant biomass Nitrification processes in soils include two pathways, i.e. [1,2]. Furthermore, transformation of nitrogen takes place in the autotrophic nitrification, and heterotrophic nitrification [7,8]. The different layers of the filter media, below the root zone, sand layer first pathway which is autotrophic nitrification occurs as a result and even in the gravel layer, which function as a biological filter. of two separate aerobic reactions, the one being the conversion of ammonia to nitrite by Ammonium-Oxidising Bacteria (AOB) These layers are important in biological processes. Nitrification that is followed by the conversion of nitrite to nitrate by Nitrite- and denitrification are the two fundamental biological processes Correspondence to: Mmasefako Lettie Mmammule Sikhosana, Department of Environmental, Water & Earth Sciences, Tshwane University of Technology, Pretoria, South Africa, Tel: (012) 382-6633; E-mail: [email protected] Received: October 26, 2020, Accepted: November 09, 2020, Published: November 16, 2020 Citation: Sikhosana MLM, Botha A, Mpenyana-Monyatsi L, Coetzee MAA (2020) Profile of Nitrification and Denitrification Bacteria in the Different Layers of Filter Media in a Rhizofiltration System. Appli Microbiol Open Access. 6:181. Copyright: © 2020 Sikhosana MLM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Appli Microbiol Open Access, Vol.6 Iss.3 No:181 1 Sikhosana MLM, et al. OPEN ACCESS Freely available online Oxidising Bacteria (NOB) [9,6]. An illustration of the conversion is oxide products are represented in (Figure 3) and specific reductase shown in (Figure 1), indicating the autotrophic chemical reaction enzymes catalyse each reduction step [26]. equation [10]. Figure 1: Autotrophic chemical reaction equation Figure 3: Intermediate gaseous nitrogen oxide products The second pathway involves heterotrophic nitrifiers, which can A large group of heterotrophic facultative anaerobic bacteria produce NO N and are capable of aerobic denitrification. Bacteria 3- performs this microbial mediated process. Since they are facultative such as Arthrobacter globiformis, Aerobacter aerogenes, Bacillus species, organisms, they can use either dissolved oxygen or nitrate as an Mycobacterium phlei, Streptomyces griseus, Thiosphaera (now known as oxygen source for metabolism and oxidation of organic matter Paracoccus denitrificans), Flavobacterium and Pseudomonas spp., as well [11,28]. However, in the presence of dissolved oxygen and nitrate, as some fungi have been found to be nitrifiers [11]. Due to the bacteria will use the dissolved oxygen first. ability of these kinds of bacteria, the heterotrophic nitrification– aerobic denitrification phenomenon was discovered to be a Denitrifying bacteria are categorised into two major groups, new way of nitrogen removal and this commonly exists in the heterotrophs and autotrophs. Heterotrophic microbes are reported natural environment and wastewater treatment facilities [12-14]. to utilize organic substrates as both carbon and energy sources. Heterotrophic nitrification of NH3 involves two proposed pathways Autotrophic nitrifiers use carbon dioxide or bicarbonate as the [15,16], whereby in the first pathway heterotrophic bacteria use carbon source, and sulphur, iron or hydrogen as energy sources similar enzymes to those used by their autotrophic counterparts [29-33]. [17]. Figure 2 illustrates the reaction process that takes place during Numerous environmental factors influence the process of heterotrophic nitrification and aerobic denitrification, the reductase denitrification. These include oxygen availability, availability in the reaction process include Ammonia MonoOxygenase (AMO), of organic matter and nitrate, pH, temperature, and degree of Hydroxylamine Oxidase (HAO), Nitrite Reductases (NIR), Nitric moisture saturation. The availability of organic matter and nitrate Oxide Reductase (NOR) and Nitrous Oxide Reductase (NOS) [18]. and the absence of oxygen are the main controlling factors [34,35]. Temperature and pH also play a role in controlling the growth of denitrifiers, metabolism, denitrification gene expression, and, subsequently, the rate of denitrification. Nitrogen removal through nitrification and denitrification has been reported to be achieved in different types of wastewater and constructed wetlands [36,37]. A previous study conducted by Wilsenach et al.investigated the application of a rhizofiltration in removing nitrogen using formulated urban effluent at Daspoort Wastewater Treatment Works; however, evidence regarding the Figure 2: The reaction process that takes place during heterotrophic presence of microbial species influencing nitrogen removal in the nitrification and aerobic denitrification filter media was not reported, despite their major role in nitrogen The second pathway appears to be limited to fungi and involves removal [38]. Information about microbial species will provide a reaction of N compounds with hydroxyl radicals formed in the engineers and designers with the knowledge to select the most presence of hydrogen peroxide and superoxide, which can occur suitable type and size of filter media to be used in the rhizofiltration during cell lysis and fungal lignin degradation [7,16]. system. The aim of the study was to identify nitrification and denitrification bacteria in different filter layers of the planted and Autotrophic nitrification is influenced by temperature, pH, unplanted sides of the system influencing nitrogen removal. alkalinity, inorganic carbon source, Dissolved Oxygen (DO), hydraulic retention time, the microbial population, and MATERIALS AND METHODS concentrations of ammonium-N [19,20]. Similarly, temperature, Site description initial pH, carbon source, C/N ratio, and DO are the major factors affecting the heterotrophic nitrification–aerobic denitrification The experimental site is located at Daspoort (Pretoria) Wastewater process. It must be noted however, that previous studies by Treatment Works. The rhizofiltration system is constructed above Patureau et al.and Su et al. have reported that the different species ground to ensure

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