bioRxiv preprint doi: https://doi.org/10.1101/397687; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 A novel function of Galactomyces candidum in highly efficient ammonia nitrogen 2 removal from low C:N wastewater 3 4 Li Xiaochao1†, Liu Hui2†, Zeng Guihua, Li Hualin2†,Chen Zhinan, Liu Wenbin1*†, 5 Yang Liping2*† 6 7 1College of Life Sciences, Hunan Normal University, Changsha, Hunan 410004, 8 China. 9 2Department of Environmental Science, Changsha Environmental Protection College, 10 Changsha, Hunan 410004, China. 11 12 *For correspondence: Liu Wenbin; Yang Liping 13 †Contributed equally 14 15 16 17 18 19 20 21 22 bioRxiv preprint doi: https://doi.org/10.1101/397687; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 23 24 Abstract 25 26 A strain of bacteria that demonstrated efficient nitrogen removal potential under low 27 C:N conditions was screened from landfill leachate. The strain was identified as 28 Galactomyces candidum by ITS sequencing, and growth density and removal of 29 ammonia nitrogen were assessed after 24 h of incubation. The results showed that the 30 optimum ammonia nitrogen reduction conditions for G. candidum was at pH 8.0 and 31 30 °C, with a C:N ratio of 1.5:1; the highest rate of ammonia nitrogen removal was 32 93.1%. This novel function of G. candidum offers great potential in the removal of 33 ammonia nitrogen from sewage, especially in low C:N wastewater. Our study 34 provides a new theoretical basis for the industrial application of bacteria in the 35 biochemical treatment of wastewater and reduces environmental pollution. 36 37 A novel function and potential of Galamyces candidum in the removal of ammonia 38 nitrogen from wastewater treatment was found,which provides a theoretical basis for 39 the treatment of ammonia in wastewater. 40 41 Keywords 42 Galactomyces candidum; low C:N; nitrogen removal; wastewater; pollution 43 mitigation 44 45 bioRxiv preprint doi: https://doi.org/10.1101/397687; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 46 47 48 Introduction 49 50 Removal of ammonia nitrogen from wastewater, especially that with low carbon 51 (C)-nitrogen (N) ratio, is of key interest (You et al., 2009; Liang et al., 2014). In 52 China, there are high levels of industrial and agricultural pollution in rivers and 53 groundwater deriving from discharge of landfill leachate, petrochemicals, fertilizers, 54 and monosodium glutamate to wastewater that is characterized by low C:N (Jia ,2014). 55 Since traditional biological treatment of wastewater requires large amounts of energy 56 and sources of carbon to complete the denitrification process ( Zhao et al.,2001), a 57 more sustainable to approach to wastewater treatment could be the identification of 58 microbes that remain effective under low carbon conditions. Fungi have different 59 carbon-nitrogen ratio requirements, where it is generally >10 (Gao et al., 2007). 60 Anammox and denitrifying bacteria have been reported to be two types of autotrophic 61 bacteria effective in low-N:N ratio of sewage (Deng et al., 2015; Persson et al., 2017), 62 while Li et al. (2017) and Yang et al. (2018) reported bacterial strains that showed 63 high ammonia nitrogen removal efficiency under low C and N conditions. 64 65 Galactomyces candidum is a eukaryotic microorganism that is widely present in the 66 environment, has morphological characteristics that are intermediate between yeast 67 and fungus, and is often used in the food and drink industry, including for cheese bioRxiv preprint doi: https://doi.org/10.1101/397687; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 68 ripening (Sacristan et al., 2012) and production of beer (Sun.2009), white wine, and 69 fermented bean curd, due to the production of pectinase (S.F. Cavalitto, et al., 2006), 70 coenzyme Q, lipase, increase medium chain fatty acid content and antimicrobial 71 activity (Khoramnia, et al., 2013). Although G. candidum has been reported to reduce 72 the harmful effects of chemical oxygen demand (COD) in sewage treatment of 73 wastewater( He, et al.,2007 ; Yan, et al.,2002; Xiong, et al.,2008), its impact on the 74 toxic pollutant, ammoniacal nitrogen in landfill leachate is unclear. Here, we screened 75 for strains of G. candidum that thrive under low C:N conditions and demonstrated 76 significant effects on ammonia removal from landfill leachate. 77 78 Results and discussion 79 80 Colony morphology 81 82 We cultured ammonia nitrogen-reducing strains of G. candidum (GC) from landfill 83 leachate in selective medium with NH4+-N as the source of N source. The colony 84 cultured on solid YPD medium at 30 °C was gray-white in color, with a diameter of c. 85 2 mm, and rough, moist surface, unraised center, and irregular edges (Fig. 1A). 86 87 Strain identification 88 89 The GC genome size was approximately 20 kb (Fig. 1B), and ITS sequence lengths of bioRxiv preprint doi: https://doi.org/10.1101/397687; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 90 two strains were about 350 and 346 bp (Fig. 1C). BLAST alignment analysis showed 91 similarity between GC and GC strain WM 04.493 KP132255.1 was 99%, and 98% 92 similarity to GC strain LC317625.1. A phylogenetic tree (Kondo et al., 2012) of GC 93 strains (Fig. 2) showed differences between GC bacteria and GC strain WM 04.493 94 KP132255.1 occurred at bases 4, 42, 43, 319, 338, 339, and 340, while differences 95 with GC strain LC317625.1 occurred at bases 65, 91, 92, 319, 338, 339, and 340 (Fig. 96 3). 97 98 Fig. 1 here. 99 100 Fig. 2 here. 101 102 Fig. 3 here. 103 104 GC growth 105 106 Growth of G. candidum strains was assessed at OD600; the initial delay in growth 107 between 0 and 2 h was followed by logarithmic growth (Fig. 4). This growth pattern 108 showed that the nitrifying bacteria had the characteristics of a short generation cycle 109 with a fast growth rate[4]. After 24 h, growth rate was stable. Since turbidity of the 110 bacteria was measured using an ultraviolet spectrophotometer, dead bacteria cells 111 were also counted, and may explain the lack of decline after the stabilization period. bioRxiv preprint doi: https://doi.org/10.1101/397687; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 112 113 Fig. 4 here. 114 + 115 Effect of initial pH on NH 4 -N removal 116 117 The optimum pH for growth of G. candidum was about 9.0 (Fig. 5A), but ammonia 118 removal efficiency was at pH. 8.0 (Fig. 5B), possibly because the ammonia reducing 119 enzymes in G. candidum are inhibited at high alkaline conditions. 120 121 Fig. 5 here. 122 + 123 Effect of temperature on NH 4 -N removal 124 125 While G. candidum grew at temperatures in the range 10-42 °C, growth was slow at 126 10 °C and more rapid at temperatures >10 °C (Fig. 6A). We found that the optimum 127 temperature for growth of the bacteria was 30 °C, and growth efficiency decreased 128 when the temperature exceeded 30 °C. Ammonia nitrogen removal rate was highest at 129 30 °C (76.3%), however this rate reduced to 7% at 42 °C (Fig. 6B). It can be seen that, 130 under different temperature conditions, the optimal growth temperature and ammonia 131 nitrogen removal rate of this strain is 30 °C. 132 133 Fig. 6 here. bioRxiv preprint doi: https://doi.org/10.1101/397687; this version posted August 22, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 134 + 135 Effect of C:N ratio on NH 4 -N removal 136 137 selection mediumWhen the C:N ratio was low, growth of G. candidum was similarly 138 low, but increased as the C increased relative to N (Fig. 7A). Ammonia nitrogen 139 removal rate was similarly low at low C:N ratios (Fig. 7B), possibly as a result of the 140 production of ammonia oxidase by the bacteria that were shown to be a strain of 141 ammoxidation heterotrophs.