Iron oxide minerals: promising materials for sustainable rice production via stimulating iron-reducing diazotrophs.

In rice paddy soil, biological nitrogen fixation is important for sustaining soil nitrogen fertility and rice growth. Anaeromyxobacter and Geobacteriaceae, iron-reducing bacteria belonging to Deltaproteobacteria, are newly discovered nitrogen-fixing bacteria dominant in paddy soils. They utilize acetate, a straw-derived major carbon compound in paddy soil, as a carbon and energy source, and ferric iron compounds as electron acceptors for anaerobic respiration. In our previous paddy field experiments, a significant increase in soil nitrogen-fixing activity was observed after the application of iron powder to straw-returned paddy field soil. In addition, combining iron application with 60–80% of the conventional nitrogen fertilizer rate could maintain rice yields similar to those with the conventional nitrogen fertilization rate. It was thus suggested that iron application to paddy soil increased the amount of nitrogen fixed in the soil by enhancing nitrogen fixation by diazotrophic iron-reducing bacteria. The present study was conducted to directly verify this suggestion by 15N-IRMS analysis combined with 15N-DNA-stable isotope probing of iron-applied and no-iron-applied plot soils in an experimental paddy field. In no-iron-applied native paddy soil, atmospheric 15N2 was incorporated into the soil by biological nitrogen fixation, in which diazotrophic iron-reducing bacteria were the most active drivers of nitrogen fixation. In iron-applied paddy soil, the amount of 15N incorporated into the soil was significantly higher due to enhanced biological nitrogen fixation, especially via diazotrophic iron-reducing bacteria, the most active drivers of nitrogen fixation in the soil. Thus, our previous suggestion was verified. This study provided a novel picture of active nitrogen-fixing microorganisms dominated by diazotrophic iron-reducing bacteria in paddy soil, and directly proved the effectiveness of iron application to enhance their nitrogen fixation and increase the incorporation of atmospheric nitrogen into soil. The enhancement of biological nitrogen fixation in paddy fields by iron application may lead to novel and unique paddy soil management strategies to increase soil nitrogen fertility and ensure rice yields with reduced nitrogen fertilizer input and lower environmental nitrogen burdens.

Biological nitrogen fixation (BNF) is important to sustain nitrogen fertility of paddy soil and rice yield, while could be affected by nitrogen fertilization. Iron-reducing bacteria, Anaeromyxobacter and Geobacter, are newly found diazotrophic bacteria predominant in paddy soil. Experimental field of this study is a long-term (35 years) nitrogen fertilized (6.0 g N/m2/year) and unfertilized paddy field, where ca. 70% of rice yield was obtained yearly in nitrogen unfertilized plot (443 ± 37 g/m2) compared to fertilized plot (642 ± 64 g/m2). Effects of long-term nitrogen fertilization/unfertilization on soil properties related to BNF were investigated with special reference to diazotrophic iron-reducing bacteria. Soil chemical/biochemical properties, soil nitrogen-fixing activity, and community composition of diazotrophic bacteria were similar between nitrogen fertilized and unfertilized plot soils. In both plot soils, Anaeromyxobacter and Geobacter were the most predominant diazotrophs. Their nifD transcripts were detected at similar level, while those of other general diazotrophs were under detection limit. It was concluded that long-term use/unuse of nitrogen fertilizer in this field did not affect the predominance and nitrogen-fixing activity of diazotrophic iron-reducing bacteria, composition of other general diazotrophs, and the resulting soil nitrogen-fixing activity. BNF, primarily driven by diazotrophic iron-reducing bacteria, might significantly contribute to sustain soil nitrogen fertility and rice yield in both plot soils. Appropriate soil management to maintain BNF, including diazotrophic iron-reducing bacteria, will be important for sustainable soil nitrogen fertility and rice production.

Effects of biofertilizers on nonsymbiotic nitrogen fixation in different paddy soils.

Currently, inorganic nitrogen fertilizer becomes a serious threat to the environment and human health. Thus, finding of alternate source of nitrogen is a viable option in assuring of sustainable agricultural system. Biological nitrogen fixation is a critical and key process in sustainable agricultural systems in tropical soils, which are frequently deficient in N and susceptible to leaching of plant nutrients. This process transforms atmospheric nitrogen to ammonia, nitrate and nitrogen dioxide. Several key abiotic and biotic factors limit legume productivity and biological nitrogen fixation in world agriculture, especially in sub-Saharan Africa. Within the soil, rhizobia frequently encounter various stresses that affect their growth, their initial steps of symbiosis and the capability of nitrogen fixation. Biotic and abiotic stresses impose a major threat to agriculture and symbiotic nitrogen fixation is dependent on host cultivar and rhizobia, but as well may be limited by pedoclimatic factors. The most common factors affecting biological nitrogen fixation and symbiosis activity in western parts of Ethiopia are soil acidity, quality of inoculants and low soil fertility. In most cases, the microsymbiont is the more affected partner, with plants growing on mineral N usually less sensitive to these stresses. Thus, it can be concluded that, particularly in a western part of Ethiopia, many studies should be focused on acidity related constraints on biological nitrogen fixation, screening of acid tolerant inoculants and low soil fertility improvements to enhance biological nitrogen fixation in smallholder farming system. Key words: Abiotic and biotic factors, legume, Rhizobium, symbiosis.

Paddy fields, as the largest anthropogenic wetlands on Earth, face a high risk of micronutrient loss through surface runoff and leaching due to their frequent irrigation-drainage cycles, as well as removal with crop harvest. While micronutrient’s losses largely impede biological nitrogen fixation (BNF) in soils, agricultural practices and mechanisms that retain micronutrients and thus increase BNF in paddy soils remain underexplored. Here we showed that the long-term (40 years) application of fertilizers increased the contents of microbial necromass in paddy soils by 20%–43% compared to the unfertilized control. In particular, long-term organic fertilizations increased poorly crystalline minerals through intensifying mineral weathering, which further contributed to the increased stable carbon burials in paddy soils. Synchrotron radiation based spectromicroscopy analysis provided direct evidence demonstrating a differential control of hydroxyl functional groups from mineral surfaces on C functional groups at the submicron scale in paddy soils. Notably, microbial necromass and short-range ordered minerals had a strong correlation with the content and bioavailability of micronutrients in paddy soils, indicating their key roles in the retention of micronutrients. Metagenomic sequencing analysis further indicated that the content and bioavailability of micronutrients were strongly correlated with the abundance of the key N-fixing genera (i.e., Azospirillum and Bradyrhizobium). Unexpectedly, structural equation modeling (SEM) identified that microbial necromass exerted the strongest control on N-fixing genera, highlighting an underappreciated role of microbial necromass as a reservoir of micronutrients. Based on micronutrient's bioavailability and metagenomic sequencing, we conclude that micronutrients are the key factor for BNF in paddy soils, offering significant implications for managing BNF in paddy soils.

Biological nitrogen fixation in rice paddy soils is driven by multiple edaphic factors and available phosphorus is the greatest contributor

Warming increased the promotion of atmospheric CO2 concentration on biological nitrogen fixation by changing the nifH gene community.

Effective utilization of the biological nitrogen fixation (BNF) activity is important for enhancing the N fertility of paddy soils and for developing sustainable rice cultivation systems. To analyze the soil factors that affect BNF in paddy soils, in this study, the effects of the temperature, water regime, and long-term soil management on 1 5 N 2 fixation were examined in relation to the decomposition of organic matter in incubation experiments. Within the range of 15-30°C, heterotrophic 1 5 N 2 fixation under dark conditions changed almost proportionally to the formation of CO 2 +CH 4 with glucose and straw as C sources by increasing the temperature, while the C use efficiency (N 2 fixed/(CO 2 +CH 4 )) was relatively higher at low temperatures in the presence of cellulose. The examination of the effect of the water regime on heterotrophic 1 5 N 2 fixation indicated that flooding after aerobic conditions promoted heterotrophic 1 5 N 2 fixation as well as the decomposition of cellulose. Among the soils with different types of management, the soils amended with manure and rice straw. showed the largest photodependent 1 5 N 2 fixation. On the other hand, the soils with a lower content of mineralizable-N tended to depend more on heterotrophic 1 5 N 2 fixation. Soils from paddy fields into upland fields (hereafter referred to as converted upland soils), particularly showed a high heterotrophic 1 5 N 2 fixation. Overall, it appeared that the management of organic matter application and the water regime may result in significant variations in BNF in paddy soils. The results obtained suggested that further studies should he conducted on the C and N metabolism involved in BNF during the decomposition processes of organic matter along with the changes in the soil redox status to identify methods for efficient soil management to promote BNF.

Nitrogen fixing nodules are formed on the roots and stems of the tropical legume Sesbania rostrata by Azorhizobium caulinodans as a result of crack entry invasion of emerging lateral roots. Advantage was taken of this invasion capability of A. caulinodans to determine whether inoculation of the non–legume wheat with A. caulinodans would result in the endophytic establishment of azorhizobia within wheat roots. Advantage was also taken of the oxygen tolerance of the nitrogenase of free–living azorhizobia to assess the extent to which the endophytic establishment of azorhizobia in wheat roots would provide a niche for nitrogen fixation of benefit to the plant. Wheat was inoculated with A. caulinodans and grown in pots under controlled conditions, without added growth reglators and without addition of fixed nitrogen. Microscopic examination of the short lateral roots of inocluated wheat showed invasion of azorhizobia between cells of the cortex, within the xylem and the root meristem Acetylene reduction assays combined with analysis of tissue nitrogen levels indicated the likelihood that colonization led to nitrogenase activity. Inoculated wheat showed significant increases in dry weight and nitrogen content as compared with uninoculated controls. We discuss the extent to which this nitrogen fixation is likely to involve symbiotic nitrogen fixation, and we indicate the need for field trials to determine the extent to which inolculation of wheat with A. caulinodans will reduce the requirement for inputs of nitrogenous fertilizers.

Biological nitrogen fixation (BNF) by methanotrophic bacteria has been shown to play an important role in maintaining fertility. However, this process is still limited to aerobic methane oxidation with sufficient oxygen. It has remained unknown whether and how methanotrophic BNF proceeds in hypoxic environments. Herein, we incubated paddy soils with a ferrihydrite-containing mineral salt medium to enrich methanotrophic bacteria in the presence of methane (20%, v/v) under oxygen constraints (0.27%, v/v). The resulting microcosms showed that ferrihydrite-dependent aerobic methane oxidation significantly contributed (81%) to total BNF, increasing the 15N fixation rate by 13-fold from 0.02 to 0.28μmol 15N2 (g dry weight soil) -1 d-1. BNF was reduced by 97% when ferrihydrite was omitted, demonstrating the involvement of ferrihydrite in methanotrophic BNF. DNA stable-isotope probing indicated that Methylocystis, Methylophilaceae, and Methylomicrobium were the dominant methanotrophs/methylotrophs that assimilated labeled isotopes (13C or 15N) into biomass. Metagenomic binning combined with electrochemical analysis suggested that Methylocystis and Methylophilaceae had the potential to perform methane-induced BNF and likely utilized riboflavin and c-type cytochromes as electron carriers for ferrihydrite reduction. It was concluded that ferrihydrite mediated methanotrophic BNF by methanotrophs/methylotrophs solely or in conjunction with iron-reducing bacteria. Overall, this study revealed a previously overlooked yet pronounced coupling of iron-dependent aerobic methane oxidation to BNF and improves our understanding of methanotrophic BNF in hypoxic zones.

Enhancement of nitrogen fixation and diazotrophs by long-term polychlorinated biphenyl contamination in paddy soil

Anaeromyxobacter and Geobacter, iron-reducing bacteria belonging to Deltaproteobacteria, are newly discovered nitrogen-fixing bacteria predominant in paddy soils. We hypothesized that adding ferric iron oxide as an electron acceptor for respiration of the iron-reducing bacteria could enhance the nitrogen-fixing activity of Anaeromyxobacter and Geobacter in paddy soils. In the soil microcosm study, soil nitrogen-fixing activity significantly increased after adding ferrihydrite or Fe2O3, ferric iron oxides, to the soil. In these soils, gene transcripts of nifD from Anaeromyxobacter and Geobacter were detected, whereas those from other general diazotrophs were not detected. Anaeromyxobacter and Geobacter might be involved in the enhanced soil nitrogen-fixing activity. The application of iron powder to paddy field soils also enhanced the soil nitrogen-fixing activity. This study is the first to demonstrate that addition of iron compounds to paddy soil enhanced the soil nitrogen-fixing activity. The results of this study may lead to the development of novel paddy soil management strategies to increase soil nitrogen fertility and ensure rice yields with a reduced nitrogen fertilizer input and a lower environmental nitrogen burden.

Organic carbon accumulation on soil mineral surfaces in paddy soils derived from tidal wetlands

Contrasting effects of iron oxides on soil organic carbon accumulation in paddy and upland fields under long-term fertilization

The study aim was to evaluate the potential nitrogen fixation and denitrification in the rhizosphere soil of potato plants, crop yield and output quality in response to the different fertilization systems and the inoculation with Azospirillum brasilense 410. Field stationary experiment was conducted between 2016 and 2019 with potato in a crop rotation system on leached chernozem soil. Farmyard manure, 40 t/ha, applied prior to potatoes planting promotes nitrogen fixation (0.8–2.0 times compared to control). However, it has also affected denitrification (in 1.4–2.2 times higher compared to control). The lowest rate of mineral fertilizers used in the experiment, N40P40K40, was shown as most environmentally feasible. Under its use the increase of soil nitrogenase activity and low denitrification levels were observed. Same trends were also noted for the medium fertilizer rate, N80P80K80. The highest doses of mineral fertilizers, N120P120K120, substantially affected the denitrification process and reduced the nitrogen fixation activity (in 1.9–2.2 times). The combination of manure with the medium fertilizers rate has also resulted in high denitrification levels, while the soil nitrogen fixation activity has restored only at flowering stage. Crop inoculation with A. brasilense combined with the manure application, has not affected studied processes. However, crop inoculation after the green manure intercropping has shown the growth of nitrogenase activity. Used on the mineral fertilizers background inoculation has activated nitrogen fixation and has ensured the decrease of denitrification levels, subject to the fertilization background. High fertilizer rates have hampered the inoculation efficiency. Inoculation has promoted crop yields on unfertilized and mineral backgrounds or following green manure. Crop inoculation following organic and the organo-mineral backgrounds had no significant effect, probably due to the competition for A. brasilense from microorganisms that have created a competitive environment for A. brasilense. Despite its environmental expediency, inoculation combined with the low fertilizer doses underperforms the action of inoculation combined with the medium fertilizer rates showing the latter as the compromise between the environmental requirements and crop productivity. The use of inoculation has promoted the accumulation of starch and ascorbic acid and has contributed to the reduction of nitrate contents in the tubers of inoculated plants.