Vinasse is a byproduct of ethanol from the sugarcane industry, generated during the distillation process (1, 2), with a production rate of 10–18 L while preparing each liter of ethanol (3). Its chemical composition varies depending on the mill plant used for the production of ethanol and the distillation process. In general, sugarcane vinasse is composed of water (93%) and organic solids and minerals (7%), such as potassium (K), calcium (Ca), and magnesium (Mg) (4, 5). This residue from ethanol production has high levels of organic matter and low C:N ratio (6) and low contents of nitrogen (N) (0.97–4.75 g L⁻¹) and phosphorus (P) (1–190 mg L⁻¹). The main non-aqueous components in vinasse are organic matter, such as glycerol, organic acids, and yeasts (5), and the volatile solids are present at high levels (6). When applied to the soil, the amount of organic material, nitrate (NO3-), ammonium (NH4+), potassium (K⁺), calcium (Ca⁺²), magnesium (Mg⁺²), sodium (Na⁺), and metals increase (5, 6). An acidic pH (3.9–5.1), high chemical oxygen demand (COD) (50.000–95.000 mg L⁻¹), and biological oxygen demand (DBO) (18.900–78.300 mg L⁻¹) also occur (6). If vinasse is poured into the environment in an unplanned way, it can cause pollution in the soil surface and underground due to the excessive accumulation of salts, which results in a toxic condition (6).

Vinasse use as a liquid fertilizer can be applied with mineral N sources in the sugarcane crops to minimize the ecological problem of its residue disposal into the environment (7, 8). However, even though this practice increases sugarcane productivity, it also causes physical, chemical, and biochemical changes in the soil environment (9, 10). There is a lack of information about the impacts of this agricultural management on the structure and functionality of soil microbial communities in tropical soils, but it is known that their changes are often correlated with soil chemical factors (11–16). This has been demonstrated for Bacteria (17–19) but not for Archaea.

Studies have shown that the addition of plant-derived organic residues commonly used in the fallow season, such as vinasse or sugarcane straw (20), increase the emissions of nitrous oxide (N₂O), a greenhouse gas (GHG) (20, 21), especially when the vinasse is combined with N addition (20, 21). Lourenço et al. (21) concluded that the amoA gene from ammonia-oxidizing Bacteria (AOB), active in the nitrification process, was the most functional gene related to N₂O emissions in sugarcane crops under vinasse fertilization. Regarding the ammonia-oxidizing Archaea (AOA), which also have amoA (22), some studies have demonstrated their relevant role in the N biogeochemical cycle (23, 24). However, experts warn about the imminent need to associate genomic data from molecular methods in microbial ecology with biochemical data to close the “gap” between physiological mechanisms by AOA and N metabolism (22).

Cloutier et al. (25) described the parallel quantification of N functional genes as one emerging technology that could be combined with high-throughput sequencing of microbial DNA to study N metabolism in soil. The microarray technology GeoChip v.5.0M (Agilent Technologies Inc., Santa Clara, CA, United States) has become a powerful and high-performance tool to monitor environmental processes with high-sensitivity detection of microbial functional genes (26–29). The chip contains 167,044 distinct probes belonging to different categories of genes involved in carbon (C) and N (ammonification, nitrification, fixation, among others) metabolisms (28). The probes were originated from gene sequences of microorganisms and correspond to a unique sequence of a gene family (Ex: aceA) and encode the same class of proteins. Several studies have concluded that this technique can be mainly used to link data from microbial communities to the processes and functions to which they are related (30) in a broad sense. Although recent studies have been able to obtain relevant information about the N metabolism genes in soil microbiota with molecular techniques (14, 16, 21, 31), GeoChip v.5.0M technology offers the advantage of analyzing many functional genes at once, providing a broader view of the functional response of the microbiota under a given condition (32).

In this sense, we would like to obtain a better insight into the archaeal and bacterial communities and microbially mediated environmental processes that play important role in the N cycle in sugarcane soil amended with vinasse and N mineral as fertilizers. We hypothesized that vinasse use combined with N fertilizer affects functional gene families and bacterial and archaeal taxonomic groups inhabiting sugarcane cultivated soils. Since the microbial N metabolism in soils is associated with N losses, such as GHGs (20), better understanding the functionality of Archaea and Bacteria can help mitigate this economic and environmental problems.

Although the use of urea as N fertilizer combined with vinasse is widely recommended, researchers and government agencies have carried out studies in the field and laboratories to evaluate their roles as emission sources of GHGs, such as N₂O, which results from this practice. Understanding the microbial processes involved in the production of this gas has become relevant for the sugar and alcohol industry since N losses in the sugarcane systems have environmental and economic impacts on society. Our results using GeoChip v.5.0M technology showed that nearly 87% of the functions related to the N metabolism might have been active in the first days after the addition of fertilizers. Although the correlations between the two datasets encompassing soil chemical factors and gene families have been limited to P and various gene families in the T7 period, at least in part due to the low number of replicate samples in this study, they suggested the occurrence of specific microbial metabolic activities in this period. The rapid increases in the availability of N and C sources in the system resulting from the addition of fertilizers (Table 1) would have led the microbiota to quickly access these less complex sources of nutrients and perform the immobilization and mineralization of N (47). Consequently, the nitrification process must have been quickly activated, which justified the great abundance of amoA genes detected in the NV treatment (Table 2). Other evidence of the occurrence of nitrification on a large scale was the increase of the ammonia monooxygenase sequences from Nitrosopumilus (AOA) (Table 3) and the reduction of the C:N ratio (Table 1) in NVT7, which suggests the possibility losses of N in the form of NO₃ besides de C (47, 48). Moreover, in the same treatment, greatest production of N₂O was detected by Navarrete et al. (19). It is known that the addition of this organic residue results in the availability of nutrients in sugarcane soils that are able to promote changes in the structure and functionality of the microbiota associated with N and C cycles (11, 19, 35).

Three nutrients analyzed here deserve to be highlighted due to their relevance in response to treatments: Zn and K showed increases, mainly in the T150 period, while P increased in the control from T7 to T150 (Table 1). A study evaluating the number of copies of the amoA gene from Archaea and Bacteria in an Australian soil submitted to different doses of Zn concluded that high doses of this nutrient could have inhibited the nitrification process of ammonia in the short term (49). In the same study, after only two years, the population of AOB became resilient to high doses of Zn and was able to perform the process. The same inhibitory effect promoted by Zn on nitrification can be observed in other studies (50, 51). Zn is one of the most abundant micronutrients and heavy metals commonly found in vinasse (52, 53) and, in our study, it was the soil chemical factor that showed the greatest increase between T7 and T150 periods in the NV treatment (Table 1). High levels of this micronutrient in the NV treatment could justify the reduction of ammonia monooxygenase in T150 associated with the amoA gene from Nitrosopumilus. In addition, it is possible that ammonia monooxygenase sequences from Nitrosomonas (AOB) increased only in T150 because of their resilience to the Zn inhibition effect.

K was another nutrient that vinasse residue supplies in large quantities (54, 55) and was already demonstrated to be a good nitrification reducer in alkaline soils (56). In an incubation experiment with potassium thiosulfate (KTS), N₂O production decreased after 12 days of application (56). Regarding P, in agricultural alkaline soils, the high availability of this nutrient can reduce the nitrification process (57). Here, although the pH values remained between 5.2 and 5.8 (acidic), its increase in control and NV treatments in T150 in relation to T7 may favor nitrification reducer by K. In relation P, our results obtained from correlation analysis showed that the levels of this nutrient were strongly associated with gene families in the first days after the application of N fertilizers, suggesting its influence on the microbial N metabolism (Figures 1, 2). The gene family hzsA, associated with the anammox process, had the greatest abundance in NVT7 and showed positive correlations with P in this study and other agricultural soils (58). This result suggests an influence of this nutrient on the anammox process, besides that of nitrification. Once in this N process, the NO₂ is used as an electron acceptor to oxidase NH₃ due to anaerobic conditions (59), and it is possible that the higher consumption of O₂ in the first days of application of the N fertilizer with vinasse may promote oxygen-limiting conditions.

In the hypothesis concerning to the most of the nutrient mobilized by the plants between 7 and 150 days after the addition of fertilizers, the high number of correlations between P and gene families in T7 would support the high N metabolism in this period. The p450nor gene family stood out in our analysis as the most correlated gene family with soil chemical factors (Figures 1, 2). It presented the greatest prevalence in NV (p = 0.008) in the T7 period according to the GeoChip analysis (Table 2). However, considering the nitric oxide reductase function from Bacteria associated with p450nor, its prevalence was in T150 (Table 3). The p450nor gene [cytochrome P450 nitric oxide (NO) reductase (NOR)] is related to the reduction of NO₂ to N₂O (60, 61). The system is located in the mitochondria and is activated during anaerobic respiration (62), which is carried out by denitrifying fungi in agricultural soils (62–64), causing plant diseases (62, 65). In Bacteria, this denitrification gene has been demonstrated to enhance virulence against the host because it uses an alternative electron acceptor, which is favorable to their lifestyle under anaerobic conditions (66). According to our results, there were significant increases in p450nor in the NV treatment and in the T7 period and, regarding its associated functions with Mycobacterium (Table 3), in the T150 period. Suleiman et al. (11) observed increases in the sporulation of fungi concomitantly with the highest volumes of N₂O during the first days after the addition of vinasse in sugarcane soils. According to Higgins et al. (67), environments with regular N inputs impose the utilization of N-oxides by the microbiota. These observations suggest that fungi denitrification by p450nor could have occurred and increased under NVT7 conditions. Lourenço et al. (21) associated the production of N₂O in sugarcane soils under vinasse with N fertilizers with the nirK gene from denitrifying fungi; however, this study did not analyze the p450nor denitrification gene. Instead, the nirK gene seems not to be assessed here on a large scale by the microbiota in NVT7 since it had the greatest abundance in N60 and sequences of nitrite reductase in N0.

The high S availability in NV may benefit from the increase of p450nor in this treatment (Table 1), which can also intensify the denitrification process, with a reduction in sulfate for the decomposition of organic matter (68) and an increase in N₂O production (69). This observation could contribute to justify the positive correlations among the p450nor gene, OM, and S in our analyses (Figures 1, 2). According to Schlüter et al. (70), the lack of O₂ and the availability of NO₃ as an electron acceptor are basic requirements for heterotrophic denitrification in soils. It is possible that the accelerated immobilization of N is readily available to microorganisms in the first days after fertilization, while the sugarcane plantlets, which do not require large amounts of this nutrient (71), favored the nitrification processes related to amoA and denitrification related to p450nor in the NV treatment.

In this study, one of the genes that showed the greatest response to NV was hao, which encodes the oxidoreductase enzyme in the nitrification process (HAO) (72, 73). Zhao et al. (74) quantified the hao gene associated with nitrifying Bacteria in a lake that received domestic sewage. The lake regions with the highest amounts of NO₃ and NH₃ showed the highest abundance of hao, suggesting the association of this gene with the eutrophication process. Is it possible that the physicochemical conditions of the soil submitted to the application of vinasse in this period were similar to a eutrophication process such as that described in Zhao et al. (74)? Analyzing the oxidoreductase enzymes that correspond to the hao gene family, Nitrosomonas (AOB), which is related to N₂O emissions by nitrification (75), had the greatest number of sequences in NVT7. Wu et al. (76) concluded that Nitrosomonas and Nitrosopumilus tended to dominate in moderately eutrophic sediment with a significant ammonium input (2.86 mM). In addition, the higher prevalence of ammonia monooxygenase and oxidoreductase sequences in NVT7 suggests a high rate of nitrification, which would also lead to the formation of NO₃ and N₂O. Smith et al. (31) concluded that the napA gene, associated with the reduction of nitrate to nitrite in denitrification, is more important in environments with lower concentrations of NO₃. In our microarray analyses, napA was more abundant in the N60 treatment and, in the MG-RAST analysis, the nitrate reductase in N0 (Tables 2, 3), suggesting that NO₃ could be found in higher concentrations in NV.

The gene families ureC and gdh are associated with the ammonification process, in which the formation of NH₄ from the organic matter by microorganisms occurs (77). Since a significant number of correlations between ureC and gdh with P were identified, we also considered the potential effect of this element on the ammonification process. Previous studies revealed the inhibitory effect of KH₂PO₄ on ammonification (78). In our study, the contents of K increase in N60 and NV treatments at the T150 period (Table 1). This could justify the increases of ureC and gdh in N60T7 and urease and glutamate dehydrogenase from Bacteria in NVT7 and their decrease in NVT150.

The results also revealed the greatest number of nitrate and nitrite transporter functions (related to ammonification) from the genus Anaeromyxobacter, a Proteobacteria (Table 3). Due to its versatile metabolism, this genus is composed of ecologically competitive Bacteria that can survive in several ecosystems, microaerophilic or anaerobic, including agricultural soils (79). This genus can reduce NO₃ to NH₄ via NO₂ as an electron acceptor and reduce N₂O (80). A specific clade from the nosZ operon, A. dehalogenans, has a higher affinity for N₂O, suggesting that they are more competitive under the environmental levels of N₂O (80). Anaeromyxobacter type nosZ sequences were found to be the most abundant nosZ genes in agricultural soils from Illinois (USA) and appear to compose a significant portion of the N₂O-reducing community (79, 81), suggesting an important role in the N cycling. The high prevalence of this Bacteria in our soils with N fertilizer, suggests favorable conditions to their development due to the microaerophilic environment and N₂O presence. On the other hand, Archaea associated with the ammonification process was frequent in the T150 period, where the salt concentrations were higher than in T7. While the non-saline soil conditions of T7 may favor the AOA Nitrosopumilus, Archaea with organic matter recycling capabilities, such as Natronomonas (from order Halobacteriales), were found in the saline soil condition in T150 (82). Natronomonas is a haloalkaline Archaea with the capacity to survive in soils with higher salt contents (82). According to our results, the highest number of urease sequences in Archaea is from Natronomonas. Some studies have shown that urease is sensitive to low pH (83), which may justify the presence of halophiles in soils with more salts.

GeoChip v.5.0M is a highly specific, sensitive, and quantitative method based on both computational and experimental assays (28). This technology can reveal information about the functionality of the microbial community in several environments not yet analyzed through other molecular techniques (28, 32). In this study, the analyses of metagenomes combined with GeoChip v.5.0M allowed the identification of which gene families associated with the N cycle significantly responded to the physicochemical changes resulting from typical agricultural management in tropical soils. Moreover, it was possible to indicate groups of Archaea and Bacteria associated with the N process, with potential increases under higher salt contents and N₂O levels, however, in low O₂ sources. This information suggests their specific metabolic capabilities in restricted conditions. The results also call attention to the increase of the p450nor gene family in these conditions that were previously associated with the denitrification process by Fungi pathogenic to plants and Mycobacterium pathogenic to animals. Finally, the Bacteria and Archaea domains are certainly ubiquitous in soil but have developed very specialized functions depending on the physical and chemical characteristics of their environments (82).

N losses in sugarcane crop soils by management practices promote economic and environmental impacts that need attention. Our results suggest “key” genes and processes in the N cycle that may be changed due to the new composition of soil factors under vinasse and urea fertilization. Our DNA analysis from the soil samples using microarray technology and metagenome annotation revealed that the gene families related to ammonification (gdh and ureC from Bacteria and Archaea), nitrification (amoA from AOA and AOB), and denitrification (p450nor from Eukarya) are the main processes responsive to this typical fertilizer management in the first days after the application. Ammonia oxidizers and halophilic Archaea were responsive to the increases in K salts and nutrients such as P in soil under vinasse application. This common practice for cultivated sugarcane may favor an increase of Anaeromyxobacter, which is capable of reducing the GHG gas N₂O.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: Metagenomics Rapid Annotation (MG-RAST) server (http://www.metagenomics.anl.gov) under project accession Metagenomes of sugarcane soils-CENA USP and accession numbers 4582104.3 to 4582153.3.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nıivel Superior, CAPES, Brazil (Grant Number: 23038.006927/2014-35/Premium 116/2017) and Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP, Brazil (Grant Numbers: 2011/51749-6 and 2012/13321-7). MC was supported by the Conselho Nacional de Desenvolvimento Cientıifico e Tecnológico, CNPq, Brazil (Grant Number: 142146/2017-0) and CAPES (Finance Code 001). AN was supported by the FAPESP (Grant Number: 2017/03575-5). AV was supported by FAPESP (Grant Number: 2019/25931-3). DB was supported by CAPES (Finance Code 001). ST was recipient of a CNPq (Grant Number: 311008/2016-0). Publication number 7301 of the Netherlands Institute of Ecology (NIOO-KNAW).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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