Accumulation of ammonium owing to the metabolic imbalance of carbon and nitrogen might inhibit the central metabolism inMethylomonassp. ZR1

Abstract

Abstract： 12The metabolic intermediates of nitrogen source have been proved to have 13 multiple functions on the metabolism of mehthanotrophs. In this study, 14 accumulation and assimilation mechanism of the nitrate metabolic 15 intermediate ammonium in the fast growing Methylomonas sp. ZR1 was 16 analyzed. Although, nitrate salt was the best nitrogen source supporting 17 the growth of ZR1, its metabolic intermediate ammonium would 18 accumulate and inhibit ZR1. Kinetic studies indicated that accumulation 19 of NH4 + was deduced from the imbalance of nitrogen and carbon 20 metabolism. Compensation of carbon skeleton α-keto-glutaramate could 21 effectively relieve the inhibition of NH4 + to ZR1, which further approved 22 42relieve the ammonium stress according to the edible carbon source. 43Revealing the endogenous ammonium accumulation mechanism and its 44 3 metabolic adjustment effect on the central metabolism of methanotrophs, 45 was meaningful to reveal the complex coordination metabolic 46 mechanism of nitrogen and carbon in methanotrophs. 47 Introduction： 49 Methanotrophs are excellent C1 compounds assimilating microorganisms, 50 and its industrial application has a long history since 1960s. At the 51 beginning methanotrophs were used to produce single cell protein from 52 methane or methanol. Nowadays, with the development of biotechnology, 53 methanotrophs were genetic modified to produce lactic acid (Henard et 54 al., 2016; Garg et al., 2018), astaxanthin (Ye and Kelly, 2012) and 55 α-hemelune (Sonntag et al., 2015) from methane. However, the slow 56 growth nature of methanotrophs has constrained its application in 57 industrial biotechnology. 58 Efforts to optimize fermentation technologies to improve the growth rates 59 of methanotrophs on methane have been reported over the past years (Yu 60 et al., 2003; Park et al., 1991b; Shah et al., 1996; Xing et al., 2006; Han et 61 al., 2009) (Myung et al., 2016). Other factors such as the addition of 62 citrate acid (Xing et al., 2006) and adjusting the ion concentration (Leak 63 and Dalton, 1986) were also reported to have effect in improving the 64 growth rate of methanotrophs from methane. These studies indicated that 65 the growth rate of methanotrophs might be result from multi-factor 66 4 combined effects. Many studies have also proved that nitrogen sources 67 have strong effect on the growth of methanotrophs. Park et al. studied 68 several factors including the nitrogen source affected the growth of OB3b, 69 and found that nitrate depletion was responsible for the diauxic growth 70 pattern in the batch cultivation of OB3b in the bioreactor. However, its 71 growth declined much with 40 mM nitrate (Park et al., 1991). Hoefman et 72 al. found niche partitioning among methanotrophic species, with methane 73 oxidation activity responses to changes in nitrogen content being 74 dependent on the in situ methanotrophic community structure (Hoefman 75 et al., 2014). Strains have developed a complex mechanism to balance the 76 carbon and nitrogen metabolism (Commic...