Variation in microbial community profiles and their energy metabolism predictions under the influence of pure 1 and mixed fertilizer in soil microcosms 2

23 The impact of environmental perturbations (e.g., nitrogen (N), phosphorus (P), potassium (K) and rice straw (Rs)) on the dynamics of soil bacterial and archaeal community are multifactor dependent and seeks more investigation 25 concerning underlyig mechanisms. Current study was designed to establish the effect of pure and mixed fertilizers on 26 microbial community profiles in paddy soil. A short-term microcosm based experiment was established in which each 27 microcosm is amended with N as C(H 2 N) 2 O, P and K as KH 2 PO 4 , K as KCl and Rs with concentrations equivalent to 28 160 kg N ha −1 , 60 kg P ha −1 , 130 kg K ha −1 and 1% respectively. Soil pH, electrical conductivity (EC), total C (TC), 29 total nirtrogen (TN), organic matter (OM), available K (AK) and extractable P (EP) were evaluated. To understand 30 the microbial community variation in soil and to predict their metabolic functions, a high throughput sequencing 31 (HTS) approach of 16S rRNA gene along with phylogenetic investigation of communities by reconstruction of 32 unobserved states (PICRUSt) was employed and analyzed. The results showed that microbial richness and diversity 33 were increased under all amnendments compared to control. Proteobacteria , Actinobacteria and Firmicutes were 34 dominant bacterial phyla. In all amendments, regarding relative abundance, Chloroflexi , Bacteroidetes and 35 Verrucomicrobia showed positive while Actinobacteria , Acidobacteria and Gemmatimonadetes showed negative 36 trends when compared with controlled observations. Thaumarchaeota and Euryarchaeota were dominant archaeal 37 phyla and exhibited increasing and decreasing trends, respectively. The PICRUSt indicated microbial community shift 38 significantly towards amino acid, carbohydrate, energy, and lipid metabolism while less towards glycan biosynthesis, 39 synthesis of secondary metabolites, terpenoids and biodegradation. Regarding metabolism (methane metabolism), 40 most and least responsive treatments were predicted to be KP and controls, respectively. These findings enhanced our 41 understanding regarding

current study are available from the corresponding author on reasonable request. The sequencing data was submitted Introduction 6 of samples but without any supplementation. These amendments were in accordance with common rice field using the USEARCH clustering algorithm at 4% sequence divergence to remove chimeras and clusters consisting of with accession numbers SAMN14661259 to SAMN14661276.

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(98%) and decreasing trend was found for Euryarchaeota with lowest in K (1.8%) and highest in C0 (6.2%). For 217 archaeal phyla, Thaumarchaeota showed increasing, Euryarchaeota proved opposite and Crenarchaeota showed both 218 trends. For Archaeal genera, Nitrososphaera and Methanosaeta showed increasing while the rest showed both trends.

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The variation in archaeal community is also well pronounced in case of methanogens.

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The observed OTUs and α-diversity indexes like Pielou's E, Faith's PD and Shannon are shown (Table 2). Of all the 225 samples, the least OTUs were found in C0 and Cf which represent lesser microbial activity without any amendment 226 and vice versa. Pielo's E and and Shannon's indexes were greater in all samples as compared to controls without plants 227 i-e C0 and Cf while the Faith's PD was also lower in these controls as compared to all other samples.

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The PICRUSt analysis demonstrated six primary functional levels, including, metabolism, genetic information 230 processing, environmental information processing, cellular processes, organ systems, and human diseases. The 231 functional profiles of metabolism at second and third level were predicted using PICRUSt and hierarchically clustered 232 as shown in figure 5. Regarding metabolism, the highest gene counts were found for amino acid and carbohydrate 233 metabolism with lowest in sample C0, Cf and P and highest in UKRs and KP which also indicate the overall trend of population size as compared to control while Actinobacteria, Acidobacteria and Gemmatimonadetes showed negative 292 trend [10,60]. Previous studies also report more response of bacterial diversity in the presence of inorganic fertilizer 293 along with rice straw which satisfy our results for all cases except UPKRs [61-63]. One contrary finding in our current 294 study was of Verrucomicrobia, which has been reported to decrease with rice straw incorporation and increase during 295 chronic N incorporation [2,60,64]. Additionally, RDA analysis showed Actinobacteria and Nitrospirae being 296 correlated with soil physicochemical properties ( Figure 6). It also showed time zero control (C0) well separated from 297 all treatments with maximum Actinobacteria population. Since microbial diversity in soil is always multifactorial 298 dependent, competitive inhibition due to multiple fertilization may justify our results. One such example is of carrier 299 ions (chloride ions in our case from KCl). Chloride ions being a strong oxidant act as a potential biocide and have 300 been studied to obstruct nitrification even at low concentration [65,66]. This study also suggest that Bacillus does not 301 seem to be very responsive genera for almost each combination except UPRs and UPKRs which propose that rice 302 straw in combination U and P may shift the functional dynamics of Bacillus. Additionally, rice straw incorporation 303 has been reported extensively to stimulate bacterial communities in paddy soil and our results are in accordance with 304 it [2, 67, 68]. Since multiple bacterial, fungal, and archaeal phyla with various functions were operating, it cannot be 305 concluded which specific factor altered their shift in our study.

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The soil archaeal community in rice fields are reported to be more stable unless influenced by temperature or presence 307 of organic matter such as rice straw [55,69]. Our results suggested archaea (specifically methanogens) being more 308 responsive as compared to bacteria concerning community structure and metabolic functioning due to KCl