Biochar amendment reassembles microbial community in a long-term phosphorus fertilization paddy soil.

This study investigates the effect of biochar amendment on microbial community structure and soil nutrient status in paddy soil that has been fertilized for an extended period of time, shedding light on sustainable agricultural practices. A 90-day incubation period revealed that biochar amendment, as opposed to long-term fertilization, significantly influenced the physicochemical properties and microbial composition of the soil. The microcosm experiment conducted using six treatments analyzed soil samples from a long-term rice ecosystem. We employed microbial biomarkers (phospholipid fatty acids, PLFAs; isoprenoid and branched glycerol dialkyl glycerol tetraethers, iGDGTs and brGDGTs; DNA) to assess microbial biomass and community structure. Biochar addition led to a decrease in PLFA biomass (15-32%) and archaeal iGDGT abundance (14-43%), while enhancing bacterial brGDGT abundance by 15-77%. Intact biochar increased archaeal and bacterial diversity, though fungal diversity remained unchanged. However, acid-washed biochar did not result in a uniform microbial diversity response. The abundance of various microbial taxa was changed by biochar amendment, including Crenarchaeota, Proteobacteria, Nitrospira, Basidiomycota, Halobacterota, Chloroflexi, Planctomycetota, and Ascomycota. Soil NH4+-N was found as the primary environmental factor impacting the composition of archaea, bacteria, and fungus in this study. These findings imply that the addition of biochar has a quick influence on the structure and activity of microbial communities, with fungi possibly having a critical role in acid paddy soil. This study contributes valuable knowledge for developing sustainable agricultural practices that promote healthy soil ecosystems. KEY POINTS: • Biochar type and phosphorus fertilization demonstrated an interactive effect on the diversity of archaea, but no such effect was observed for bacteria and fungi. • Soil fungi contribute to approximately 20% of the total phospholipid fatty acid (PLFA) content. • Biochar, especially acid-washed rice straw biochar, increases glucose metabolism in bacteria and archaea and decreases saprophytic fungi.

Different Responses of Soil Bacterial Communities to Nitrogen Addition in Moss Crust.

Bacterial communities in soil serve an important role in controlling terrestrial biogeochemical cycles and ecosystem processes. Increased nitrogen (N) deposition in Northwest China is generating quantifiable changes in many elements of the desert environment, but the impacts of N deposition, as well as seasonal variations, on soil bacterial community composition and structure are poorly understood. We used high-throughput sequencing of bacterial 16S rRNA genes from Gurbantünggüt Desert moss crust soils to study the impacts of N addition on soil bacterial communities in March, May, and November. In November, we discovered that the OTU richness and diversity of soil bacterial community dropped linearly with increasing N input. In November and March, the diversity of the soil bacterial community decreased significantly in the medium-N and high-N treatments. In May, N addition caused a substantial change in the makeup of the soil bacterial composition, while the impacts were far less apparent in November and March. Furthermore, the relative abundance of major bacterial phyla reacted non-linearly to N addition, with high-N additions decreasing the relative richness of Proteobacteria, Bacteroidetes, and Acidobacteria while increasing the relative abundance of Actinobacteria and Chloroflexi. We also discovered that seasonality, as characterized by changes in soil moisture, pH, SOC, and AK content, had a significant impact on soil bacterial communities. Significant variations in the makeup of the community were discovered at the phylum and genus levels throughout the various months. In May, the variety of soil bacterial community was at its peak. Further investigation showed that the decrease in soil bacterial diversity was mostly attributed to a drop in soil pH. These results indicated that the impact of N deposition on the soil bacterial community was seasonally dependent, suggesting that future research should evaluate more than one sample season at the same time.

Air pollution could drive global dissemination of antibiotic resistance genes.

Antibiotic-resistant pathogens pose a significant threat to human health. Several dispersal mechanisms have been described, but transport of both microbes and antibiotic resistance genes (ARGs) via atmospheric particles has received little attention as a pathway for global dissemination. These atmospheric particles can return to the Earth's surface via rain or snowfall, and thus promote long-distance spread of ARGs. However, the diversity and abundance of ARGs in fresh snow has not been studied and their potential correlation with particulate air pollution is not well explored. Here, we characterized ARGs in 44 samples of fresh snow from major cities in China, three in North America, and one in Europe, spanning a gradient from pristine to heavily anthropogenically influenced ecosystems. High-throughput qPCR analysis of ARGs and mobile genetic elements (MGEs) provided strong indications that dissemination of ARGs in fresh snow could be exacerbated by air pollution, severely increasing the health risks of both air pollution and ARGs. We showed that snowfall did effectively spread ARGs from point sources over the Earth surface. Together our findings urge for better pollution control to reduce the risk of global dissemination of antibiotic resistance genes.

Combined genome-centric metagenomics and stable isotope probing unveils the microbial pathways of aerobic methane oxidation coupled to denitrification process under hypoxic conditions.

Obligate aerobic methanotrophs have been proven to oxidize methane and participate in denitrification under hypoxic conditions. However, this phenomenon and its metabolic mechanism have not been investigated in detail in aerobic methane oxidation coupled to denitrification (AME-D) process. In this study, a type of hypoxic AME-D consortium was enriched and operated for a long time in a CH4-cycling bioreactor with strict anaerobic control and the nitrite removal rate reached approximately 50 mg N/L/d. Metagenomics combined with DNA stable-isotope probing demonstrated that the genus Methylomonas, which constitutes type I aerobic methanotrophs, was the dominant member and contributed to methane oxidation and partial denitrification. Metagenomic binning recovered a near-complete (98%) draft genome affiliated with the family Methylococcaceae containing essential genes that encode nitrite reductase (nirK), nitric oxide reductase (norBC) and hydroxylamine dehydrogenase (hao). Metabolic reconstruction of the selected Methylococcaceae genomes also revealed a potential link between methanotrophy and partial denitrification.

Phosphorus Input Alters the Assembly of Rice (Oryza sativa L.) Root-Associated Communities.

Rice root-associated microbial community play an important role in plant nutrient acquisition, biomass production, and stress tolerance. Herein, root-associated community assembly was investigated under different phosphate input levels in phosphorus (P)-deficient paddy soil. Rice was grown in a long-term P-depleted paddy soil with 0 (P0), 50 (PL), or 200 (PH) mg P2O5 kg-1 application. DNA from root endophytes was isolated after 46 days, and PCR amplicons from archaea, bacteria, and fungi were sequenced by an Illumina Miseq PE300 platform, respectively. P application had no significant effect on rice root endophytic archaea, which were dominated by ammonia-oxidizing Candidatus Nitrososphaera. By contrast, rice root endophytic community structure of the bacteria and fungi was affected by soil P. Low P input increased endophytic bacterial diversity, whereas high P input increased rhizosphere fungi diversity. Bacillus and Pleosporales, associated with phosphate solubilization and P uptake, dominated in P0 and PH treatments, and Pseudomonas were more abundant in the PL treatment than in the P0 and PH treatments. Co-occurrence network analysis revealed a close interaction between endophytic bacteria and fungi. Soil P application affected both the rice root endosphere and soil rhizosphere microbial community and interaction between rice root endophytic bacteria, and fungi, especially species related to P cycling.

Anaerobic ammonium oxidation is a major N-sink in aquifer systems around the world.

Global-scale N-oxide contamination of groundwater within aquifers occurs due to the widespread use of N-bearing fertilizers and chemicals, threatening both human and environmental health. However, the conversion of these pollutants in active nitrogen (N) cycling processes in the subsurface biosphere still remains unclear. This study investigates the global occurrence of anaerobic ammonium oxidation (anammox) in aquifers, where anammox was found to be turned on and off between saturated and unsaturated soil horizons, and contributed 36.8-79.5% to N loss in saturated soil horizons, the remainder being due to denitrification which has traditionally been considered the main pathway for removal of N-pollutants from aquifers. Although anammox activity was undetectable in the unsaturated soil horizons, it could potentially be activated by contact with ascending groundwater. High-throughput pyrosequencing analysis identified Candidatus Brocadia anammoxidans as being the most abundant anammox bacterium in the saturated soils investigated. However, the anammox bacterial abundance was determined by the relative richness of Candidatus Jettenia asiatica. Isotopic pairing experiments revealed that coupling anammox with ammonium oxidation and respiratory ammonification enabled the formation of a revised N cycle in aquifer systems, in which respiratory ammonification acted as an important coordinator. Anammox can therefore contribute substantially to aquifer N cycling and its role in remediation of aquifers contaminated with N-oxides may be of global importance.

Microbiomes inhabiting rice roots and rhizosphere.

Land plants directly contact soil through their roots. An enormous diversity of microbes dwelling in root-associated zones, including endosphere (inside root), rhizoplane (root surface) and rhizosphere (soil surrounding the root surface), play essential roles in ecosystem functioning and plant health. Rice is a staple food that feeds over 50% of the global population. Its root is a unique niche, which is often characterized by an oxic region (e.g. the rhizosphere) surrounded by anoxic bulk soil. This oxic-anoxic interface has been recognized as a pronounced hotspot that supports dynamic biogeochemical cycles mediated by various functional microbial groups. Considering the significance of rice production upon global food security and the methane budget, novel insights into how the overall microbial community (i.e. the microbiome) of the rice root system influences ecosystem functioning is the key to improving crop health and sustainable productivity of paddy ecosystems, and alleviating methane emissions. This mini-review summarizes the current understanding of microbial diversity of rice root-associated compartments to some extent, especially the rhizosphere, and makes a comparison of rhizosphere microbial community structures between rice and other crops/plants. Moreover, this paper describes the interactions between root-related microbiomes and rice plants, and further discusses the key factors shaping the rice root-related microbiomes.

Effects of nickel and cobalt on methane production and methanogen abundance and diversity in paddy soil.

BACKGROUND: Paddies are an important anthropogenic source of methane emissions to the atmosphere, and they are impacted by heavy metal pollution. Nickel (Ni) and cobalt (Co) pollution might either enhance or mitigate CH4 emission from paddy soils due to the total amounts of metals, bioavailability and functional microbial activity and composition. METHODS: An incubation experiment was conducted, and different Ni and Co concentrations were added to test the effects of trace metals on methane production in paddy soil. The archaea community structure and the abundance of methanogen functional groups in the paddy soil with added Ni and Co were detected using high-throughput sequencing and quantitative PCR based on the 16S rRNA and mcrA (methyl coenzyme M reductase) genes, respectively. RESULTS: The highest methane production rate was 561 mg CH4 kg-1 dry soil d-1 with the addition of 50 mg kg-1 Ni and 684 mg CH4 kg-1 dry soil d-1 with the addition of 25 mg kg-1 Co. Accordingly, the mcrA gene was most abundant in the 50 mg kg-1 Ni addition (3.1 × 106 ± 0.5 × 106 copies g-1 dry soil). The lowest mcrA gene abundance was detected in the 500 mg kg-1 Co addition (9.2× 105 ±  0.4 × 105 copies g-1 dry soil). The dominant methanogens were Methanobacterium, Methanosarcina, Methanocella, Methanomassiliicoccus, Bathyarchaeota, and Rice Cluster I (RC-I), and the relative abundances of these groups were higher than 1% in the Ni and Co treatments. Additionally, the archaeal compositions differed significantly in the soils with various Ni and Co additions. The most abundant Methanococcus spp. represented 51.3% of the composition in the 50 mg kg-1 Ni addition, which was significantly higher than that of the control (12.9% to 17.5%). DISCUSSION: Our results indicated that the contamination of soil by Ni and Co significantly affected total methanogens abundance and specific methanogen functional groups. Ni and Co additions to paddy soil promoted methanogenic activity at low concentrations, while they had inhibitory effects at high concentrations. Because paddy soils largely contribute to methane emissions and are increasingly exposed to heavy metal pollution, our results show that future assessments of greenhouse gas flux from paddy soils should take into account the effects of pollution by Ni and Co.

Temporal and spatial impact of Spartina alterniflora invasion on methanogens community in Chongming Island, China.

Methane production by methanogens in wetland is recognized as a significant contributor to global warming. Spartina alterniflora (S. alterniflora), which is an invasion plant in China's wetland, was reported to have enormous effects on methane production. But studies on shifts in the methanogen community in response to S. alterniflora invasion at temporal and spatial scales in the initial invasion years are rare. Sediments derived from the invasive species S. alterniflora and the native species Phragmites australis (P. australis) in pairwise sites and an invasion chronosequence patch (4 years) were analyzed to investigate the abundance and community structure of methanogens using quantitative real-time PCR (qPCR) and Denaturing gradient gel electrophoresis (DGGE) cloning of the methyl-coenzyme M reductase A (mcrA) gene. For the pairwise sites, the abundance of methanogens in S. alterniflora soils was lower than that of P. australis soils. For the chronosequence patch, the abundance and diversity of methanogens was highest in the soil subjected to two years invasion, in which we detected some rare groups including Methanocellales and Methanococcales. These results indicated a priming effect at the initial invasion stages of S. alterniflora for microorganisms in the soil, which was also supported by the diverse root exudates. The shifts of methanogen communities after S. alterniflora invasion were due to changes in pH, salinity and sulfate. The results indicate that root exudates from S. alterniflora have a priming effect on methanogens in the initial years after invasion, and the predominate methylotrophic groups (Methanosarcinales) may adapt to the availability of diverse substrates and reflects the potential for high methane production after invasion by S. alterniflora.

Contrasting beneficial and pathogenic microbial communities across consecutive cropping fields of greenhouse strawberry.

Soil weakness across consecutive cropping fields can be partially explained by the changes in microbial community diversity and structure. Succession patterns and co-occurrence mechanisms of bacteria and fungi, especially beneficial or pathogenic memberships in continuous cropping strawberry fields and their response to edaphic factors remained unclear. In this study, Illumina sequencing of bacterial 16S ribosomal RNA and fungal internal transcribed spacer genes was applied in three time-course (1, 5, and 10 years) fields across spring and winter. Results showed that the richness and diversity of bacterial and fungal communities increased significantly (p < 0.05) in 1-year field and decreased afterwards across two seasons. Network analysis revealed beneficial bacterial and fungal genus (Bacillus and Trichoderma) dominated under 1-year field whereas Fusarium accumulated under 10-year field at either season. Moreover, Trichoderma harzianum and Bacillus subtilis that have been reported to effectively control Fusarium wilt in strawberries accumulated significantly under 1-year field. Canonical correspondence analysis showed that beneficial bacterial Rhodospirillales and Rhizobiales and fungal Glomerales accumulated in 1-year field and their distributions were significantly affected by soil pH, microbial biomass C (MBC), and moisture. On the contrary, fungal pathogenic species Fusarium oxysporum strongly increased under 10-year field at the winter sample and the abundance was positively (p < 0.01) correlated with soil moisture. Our study suggested that the potential of microcosm under 1-year field stimulates the whole microbial diversity and favors different beneficial taxa across two seasons. Soil pH, moisture, and MBC were the most important edaphic factors leading to contrasting beneficial and pathogenic memberships across consecutive strawberry cropping fields.

pH rather than nitrification and urease inhibitors determines the community of ammonia oxidizers in a vegetable soil.

Nitrification inhibitors and urease inhibitors, such as nitrapyrin and N-(n-butyl) thiophosphoric triamide (NBPT), can improve the efficiencies of nitrogen fertilizers in cropland. However, their effects on ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) across different soil pH levels are still unclear. In the present work, vegetable soils at four pH levels were tested to determine the impacts of nitrification and urease inhibitors on the nitrification activities, abundances and diversities of ammonia oxidizers at different pHs by real-time PCR, terminal restriction fragment length polymorphism (T-RFLP) and clone sequence analysis. The analyses of the abundance of ammonia oxidizers and net nitrification rate suggested that AOA was the dominate ammonia oxidizer and the key driver of nitrification in acidic soil. The relationships between pH and ammonia oxidizer abundance indicated that soil pH dominantly controlled the abundance of AOA but not that of AOB. The T-RFLP results suggested that soil pH could significantly affect the AOA and AOB community structure. Nitrapyrin decreased the net nitrification rate and inhibited the abundance of bacterial amoA genes in this vegetable soil, but exhibited no effect on that of the archaeal amoA genes. In contrast, NBPT just lagged the hydrolysis of urea and kept low NH4+-N levels in the soil at the early stage. It exhibited no or slight effects on the abundance and community structure of ammonia oxidizers. These results indicated that soil pH, rather than the application of urea, nitrapyrin and NBPT, was a critical factor influencing the abundance and community structure of AOA and AOB.

Microbial community structures and metabolic profiles response differently to physiochemical properties between three landfill cover soils.

Landfills are always the most important part of solid waste management and bear diverse metabolic activities involved in element biogeochemical cycling. There is an increasing interest in understanding the microbial community and activities in landfill cover soils. To improve our knowledge of landfill ecosystems, we determined the microbial physiological profiles and communities in three landfill cover soils (Ninghai: NH, Xiangshan: XS, and Fenghua: FH) of different ages using the MicroResp(TM), phospholipid fatty acid (PLFA), and high-throughput sequencing techniques. Both total PLFAs and glucose-induced respiration suggested more active microorganisms occurred in intermediate cover soils. Microorganisms in all landfill cover soils favored L-malic acid, ketoglutarate, and citric acid. Gram-negative bacterial PLFAs predominated in all samples with the representation of 16:1ω7, 18:1ω7, and cy19:0 in XS and NH sites. Proteobacteria dominated soil microbial phyla across different sites, soil layers, and season samples. Canonical correspondence analysis showed soil pH, dissolved organic C (DOC), As, and total nitrogen (TN) contents significantly influenced the microbial community but TN affected the microbial physiological activities in both summer and winter landfill cover soils.

Community structure and soil pH determine chemoautotrophic carbon dioxide fixation in drained paddy soils.

Previous studies suggested that microbial photosynthesis plays a potential role in paddy fields, but little is known about chemoautotrophic carbon fixers in drained paddy soils. We conducted a microcosm study using soil samples from five paddy fields to determine the environmental factors and quantify key functional microbial taxa involved in chemoautotrophic carbon fixation. We used stable isotope probing in combination with phospholipid fatty acid (PLFA) and molecular approaches. The amount of microbial (13)CO2 fixation was determined by quantification of (13)C-enriched fatty acid methyl esters and ranged from 21.28 to 72.48 ng of (13)C (g of dry soil)(-1), and the corresponding ratio (labeled PLFA-C:total PLFA-C) ranged from 0.06 to 0.49%. The amount of incorporationof (13)CO2 into PLFAs significantly increased with soil pH except at pH 7.8. PLFA and high-throughput sequencing results indicated a dominant role of Gram-negative bacteria or proteobacteria in (13)CO2 fixation. Correlation analysis indicated a significant association between microbial community structure and carbon fixation. We provide direct evidence of chemoautotrophic C fixation in soils with statistical evidence of microbial community structure regulation of inorganic carbon fixation in the paddy soil ecosystem.

Acidophilic denitrifiers dominate the N2O production in a 100-year-old tea orchard soil.

Aerobic denitrification is the main process for high N2O production in acid tea field soil. However, the biological mechanisms for the high emission are not fully understood. In this study, we examined N2O emission and denitrifier communities in 100-year-old tea soils with four pH levels (3.71, 5.11, 6.19, and 7.41) and four nitrate concentration (0, 50, 200, and 1000 mg kg(-1) of NO3 (-)-N) addition. Results showed the highest N2O emission (10.1 mg kg(-1) over 21 days) from the soil at pH 3.71 with 1000 mg kg(-1) NO3 (-) addition. The N2O reduction and denitrification enzyme activity in the acid soils (pH <7.0) were significantly higher than that of soils at pH 7.41. Moreover, TRF 78 of nirS and TRF 187 of nosZ dominated in soils of pH 3.71, suggesting an important role of acidophilic denitrifiers in N2O production and reduction. CCA analysis also showed a negative correlation between the dominant denitrifier ecotypes (nirS TRF 78, nosZ TRF 187) and soil pH. The representative sequences were identical to those of cultivated denitrifiers from acidic soils via phylogenetic tree analysis. Our results showed that the acidophilic denitrifier adaptation to the acid environment results in high N2O emission in this highly acidic tea soil.

Biogeographical distribution of denitrifying anaerobic methane oxidizing bacteria in Chinese wetland ecosystems.

The discovery of denitrifying anaerobic methane oxidation with nitrite as electron acceptor mediated by 'Candidatus Methylomirabilis oxyfera' connected the biogeochemical carbon and nitrogen cycle in a new way. However, it is important to have a comprehensive understanding about the distribution of M. oxyfera-like bacteria in the terrestrial realm, especially the wetland ecosystems that are known as the largest natural source of atmospheric methane. Here, our molecular evidence demonstrated that a wide geographical distribution of M. oxyfera-like bacteria at oxic/anoxic interfaces of various wetlands (n = 91) over the Chinese territory. Intriguingly, the M. oxyfera-like bacteria were detected in some extreme environments, indicating that M. oxyfera-like bacteria occupied a wide range of habitats. Quantitative polymerase chain reaction estimated that the abundance of M. oxyfera-like bacteria ranged from 2.2 × 10(3) to 2.3 × 10(7) copies g(-1) dry soil, and up to around 0.62% of the total number of bacteria. Moreover, the M. oxyfera-like bacteria showed high biodiversity in wetland ecosystems based on the analysis of 462 pmoA and 287 16S rRNA gene sequences. The current study revealed the widespread distribution and biogeography of M. oxyfera-like bacteria in the terrestrial system.

High abundance and diversity of nitrite-dependent anaerobic methane-oxidizing bacteria in a paddy field profile.

The discovery of nitrite-dependent anaerobic methane oxidation (n-damo) mediated by 'Candidatus Methylomirabilis oxyfera' with nitrite and methane as substrates has connected biogeochemical carbon and nitrogen cycles in a new way. The paddy fields often carry substantial methane and nitrate, thus may be a favorable habitat for n-damo bacteria. In this paper, the vertical-temporal molecular fingerprints of M. oxyfera-like bacteria, including abundance and community composition, were investigated in a paddy soil core in Jiangyin, near the Yangtze River. Through qPCR investigation, high abundance of M. oxyfera-like bacteria up to 1.0 × 10(8) copies (g d.w.s.)(-1) in summer and 8.5 × 10(7) copies (g d.w.s.)(-1) in winter was observed in the ecotone of soil and groundwater in the paddy soil core, which was the highest in natural environments to our knowledge. In the ecotone, the ratio of M. oxyfera-like bacteria to total bacteria reached peak values of 2.80% in summer and 4.41% in winter. Phylogenetic analysis showed n-damo bacteria in the paddy soil were closely related to M. oxyfera and had high diversity in the soil/groundwater ecotone. All of the results indicated the soil/groundwater ecotone of the Jiangyin paddy field was a favorable environment for the growth of n-damo bacteria.

[Contribution of fungi to soil nitrous oxide emission and their research methods: a review].

Nitrous oxide is an important greenhouse gas. Soil is one major emission source of N2O, which is a by-product of microorganisms-driven nitrification and denitrification processes. Extensive research has demonstrated archaea and bacteria are the predominant contributors in nitrification and denitrification. However, fungi may play a predominant role in the N transformation in a certain soil ecosystem. The fungal contribution to N2O production has been rarely investigated. Here, we reviewed the mechanism of N2O production by soil fungi. The mechanisms of denitrification, autotrophic and heterotrophic nitrification and their key microbes and functional genes were described, respectively. We discriminated the differences in denitrification between bacteria and fungi and discussed the methods being used to determine the contribution of fungi to soil N2O emission, including selective inhibitors, 15N stable isotope probing, isolation and pure culturing and uncultured molecular detection methods. The existing problems and research prospects were also presented.

Shifts in the abundance and community structure of soil ammonia oxidizers in a wet sclerophyll forest under long-term prescribed burning.

Fire shapes global biome distribution and promotes the terrestrial biogeochemical cycles. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) play a vital role in the biogeochemical cycling of nitrogen (N). However, behaviors of AOB and AOA under long-term prescribed burning remain unclear. This study was to examine how fire affected the abundances and communities of soil AOB and AOA. A long-term repeated forest fire experiment with three burning treatments (never burnt, B0; biennially burnt, B2; and quadrennially burnt, B4) was used in this study. The abundances and community structure of soil AOB and AOA were determined using quantitative PCR, restriction fragment length polymorphism and clone library. More frequent fires (B2) increased the abundance of bacterium amoA gene, but tended to decrease archaeal amoA genes. Fire also modified the composition of AOA and AOB communities. Canonical correspondence analysis showed soil pH and dissolved organic C (DOC) strongly affected AOB genotypes, while nitrate-N and DOC shaped the AOA distribution. The increased abundance of bacterium amoA gene by fires may imply an important role of AOB in nitrification in fire-affected soils. The fire-induced shift in the community composition of AOB and AOA demonstrates that fire can disturb nutrient cycles.