Piggery wastewater treatment by Acinetobacter sp. TX5 immobilized with spent mushroom substrate in a fixed-bed reactor
Graphical abstract
Introduction
As a high strength wastewater, piggery wastewater usually contains nitrogen, phosphorus and other hazardous compounds. Inadequate treatment and uncontrolled discharge of this wastewater can lead to epidemic diseases and unpleasant odors (Zhao et al., 2014), which necessitates the application of appropriate remediation technologies. Though some physical and chemical treatment methods have been proposed and proved to be effective, the biological process is less expensive and drawing extensive attention. More specifically, microbial technics seem to be more suitable for the treatment of piggery wastewater since there are a variety of nutrients (Ganeshkumar et al., 2018) and trace elements (Meng et al., 2018) in wastewater that are all propitious for the growth of microorganisms. Meanwhile, to improve the removal efficiency of pollutants, immobilized cells technology has been increasingly studied in recent years (Kesseru et al., 2003; Ma et al., 2015; Song et al., 2005), and various matrices were used for the immobilization of denitrifiers such as activated carbon, calcium alginate, agar, glass beads and sodium alginate (Long et al., 2004). Also, the immobilization technology was applied on the piggery wastewater treatment (Lin et al., 2006; Su et al., 2014; Su et al., 2001). Nevertheless, the above matrices could result in a relatively high cost when used in the engineering practice, and therefore a much cheaper matrix would be required.
As an agricultural waste, spent mushroom substrates (SMSs) are produced in large quantities every year in the world, which has incurred a serious problem to the local environment. Consequently, to reuse these materials as well as gain economic benefit, SMSs have been explored in various fields like environmental remediation (Garcia-Delgado et al., 2015; Jia et al., 2017; Wang et al., 2016; Zang et al., 2017), enzymes production (Yang et al., 2015) and organic fertilizers (Sochtig and Grabbe, 1995). Out of them, environmental remediation by SMSs was most studied as they usually have a good capacity to adsorb pollutants owing to a large surface area that results from the degradation by fungi. This also means that SMSs could be used as immobilization materials on which microorganisms can attach and grow. To our knowledge, however, no research on immobilizing cells with SMSs has been reported so far.
Various heterotrophic microorganisms were found to be able to perform simultaneous nitrification and denitrification, among which Acinetobacter genus was demonstrated to possess a good capability of nitrogen removal (Sarioglu et al., 2012; Su et al., 2017b; Su et al., 2015c). Likewise, in order to achieve enhanced nitrogen removal by this genus in aquatic environments, immobilization technique was applied on Acinetobacter calcoaceticus STB1 (Sarioglu et al., 2013), Acinetobacter sp. Y1 (Lv et al., 2013) and Acinetobacter sp. J25 (Su et al., 2017a), indicating that Acinetobacter genus has a good prospect for the engineering application. In the meantime, researchers have been attempting to figure out the mechanism of nitrogen removal in this genus. From the genetic level, the napA gene (Huang et al., 2013) and nirS gene (Su et al., 2015b) were identified to confirm the aerobic denitrification. By comparison, functional enzymes involved in nitrogen removal appeared to be more complicated as nitrate and nitrite reductases could be detected in some strains (Liu et al., 2015; Ren et al., 2014; Su et al., 2015a) but not detected in Acinetobacter calcoaceticus HNR (Zhao et al., 2010). Although two important enzymes, ammonia monooxygenase (AMO) (Zhang et al., 2015) and hydroxylamine oxidase (HAO) (Zhang et al., 2014), have been well purified from Acinetobacter sp. Y16, other key enzymes like denitrifying nitrite reductase (dNiR) has not been isolated from this genus so far, and thus the corresponding mechanism for nitrogen removal is still not very clear.
In the current study, Acinetobacter sp. TX5 with a good ability to conduct heterotrophic nitrification and aerobic denitrification was isolated from a sequencing batch reactor (SBR) treating municipal wastewater. TX5 cells were subsequently immobilized with SMSs and packed into a fixed-bed reactor to continuously treat piggery wastewater. To clarify the mechanism of nitrogen removal in TX5, the isotope analysis, purification of a denitrifying nitrite reductase (dNiR) and enzymatic properties analysis were carried out. Moreover, the nitrogen removal pathway in Acinetobacter sp. TX5 was proposed.
Section snippets
Medium and piggery wastewater
The screening medium (SM) was composed of (g/L): sodium succinate 8.6, KNO3 1.0, KH2PO4 1.0, FeCl2·6H2O 0.05, CaCl2·2H2O 0.02, MgSO4·7H2O 1.2, pH 7.0–7.2. Bromothymol Blue (BTB) medium contained the following ingredients (g/L): sodium succinate 8.5, KNO3 1.0, KH2PO4 1.0, FeCl2·6H2O 0.5, CaCl2·2H2O 0.2, MgSO4·7H2O 1.2, BTB (1% alcoholic solution) 1 mL, agar (for solid) 20, pH 7.0–7.2. Luria-Bertani (LB) medium comprised (g/L): NaCl 10, Tryptone 10, Yeast extract 5, pH 7.0–7.2. The simultaneous
Isolation and identification of strains
In this study, five blue strains on BTB plate were selected and respectively inoculated into SNDM to investigate the capability of nitrogen removal under aerobic conditions, among which the strain TX5 displayed the highest efficiency (data not shown) to remove nitrogen and thus was used for identification. Based on 16S rRNA genes, the phylogenetic tree was constructed (Fig. S3), from which TX5 presented a 99% genetic homology with Acinetobacter sp. and therefore was identified as Acinetobacter
Conclusions
Acinetobacter sp. TX5 capable of nitrogen removal was immobilized with SHMS to treat the real RPW. The following conclusions can be drawn:
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TX5 cells immobilized with SHMS were comparable to those immobilized with activated carbon when used for the RPW treatment.
- 2)
SHMS-immobilized beads could adapt to the appropriately diluted RPW rapidly and have a good capability to treat RPW continuously in a fixed-bed reactor, and thus they would be more suitable for the engineering practice.
- 3)
The nitrogen
Acknowledgements
This work was supported by the Natural Science Foundation of Fujian Province (no. 2015J01594) and the National Natural Science Foundation of China (no. 21407024).
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These authors contributed equally to this work.