Widespread bacterial responses and their mechanism of bacterial metallogenic detoxification under high concentrations of heavy metals.

Microbial mineralization is increasingly used in bioremediation of heavy metal pollution, but better mechanistic understanding of the processes involved and how they are regulated are required to improve the practical application of microorganisms in bioremediation. We used a combination of morphological (TEM) and analytical (XRD, XPS, FTIR) methods, together with novel proteomic analyses, to investigate the detoxification mechanisms, used by a range of bacteria, including the strains Bacillus velezensis LB002, Escherichia coli DH5α, B. subtilis 168, Pseudomonas putida KT2440, and B. licheniformis MT-1, exposed to elevated concentrations of Cd2+ and combinations of Cd2+, Pb2+, Cu2+, and Zn2+, in the presence and absence of added CaCl2. Common features of detoxification included biomineralization, including the production of biological vaterite, up-regulation of proteins involved in flagellar movement and chemotaxis, biofilm synthesis, transmembrane transport of small molecules and organic matter decomposition. The putative roles of differentially expressed proteins in detoxification are discussed in relation to chemical and morphological data and together provide important tools to improve screening, selection, and practical application of bacterial isolates in bioremediation of polluted environments.

EIN3 and PIF3 Form an Interdependent Module That Represses Chloroplast Development in Buried Seedlings.

In buried seedlings, chloroplasts are arrested at the etioplast stage, but they rapidly mature upon emergence of the seedling. Etioplast-chloroplast differentiation is halted through the integration of soil-induced signals, including pressure and the absence of light, although the details on how this information converges to regulate cellular decisions remain unclear. Here, we identify an interdependent transcription module that integrates the mechanical pressure and darkness signals to control chloroplast development in Arabidopsis thaliana Mutations of ETHYLENE-INSENSITIVE3 (EIN3), the primary transcription factor in the ethylene signaling pathway that is activated in response to mechanical pressure, cause early development of etioplasts in the dark and severe photobleaching upon light exposure. Genetic studies demonstrate that repression of etioplast differentiation by EIN3 requires PHYTOCHROME INTERACTING FACTOR3 (PIF3), a darkness-stabilized bHLH transcription factor. EIN3 and PIF3 directly interact and form an interdependent module to repress the expression of most LIGHT HARVESTING COMPLEX (LHC) genes; overexpressing even one LHC could cause premature development of etioplasts. The EIN3-PIF3 transcription module synergistically halts chloroplast development by interdependently co-occupying the promoters of LHC genes. Thus, our results define a transcriptional regulatory module and provide mechanistic insight on the concerted regulation of chloroplast development by multiple soil-induced signals.

Effects of simulated acid rain on hydrochemical factors and microbial community structure in red soil aquifers.

Acid rain can lower the pH of groundwater and affect its hydrogeochemistry and microbial ecology. However, the effects of acid rain on the hydrogeochemistry and microbial ecology of red soil groundwater systems in southern China are poorly understood. Previous research had mainly investigated the sources and patterns of groundwater acidification, but not the microbial mechanisms that contribute to this process and their associations with hydrochemical factors. To address this knowledge gap, we conducted a soil column experiment to simulate the infiltration of acid rain through various filter materials (coarse, medium, and fine sand) and to examine the hydrochemical and microbial features of the infiltrate, which can reveal how simulated acid rain (pH 3.5-7.0) alters the hydrochemistry and microbial community composition in red soil aquifers. The results showed that the pH of the leachate decreased due to simulated acid rain, and that the leaching efficiency of nitrogen and metal ions was influenced by the particle size of the filter media. Illumina 16S rRNA gene sequencing revealed that the leachate was dominated by Proteobacteria, Patescibacteria, Actinobacteria, and Acidobacteria, with Proteobacteria accounting for 67.04-74.69% of the bacterial community and containing a high proportion of nitrifying and denitrifying bacteria. Additionally, several genera with heavy metal tolerance, such as Burkholderia-Caballeronia-Paraburkholderia, Delftia, Methylversatilis, Aquicella, and Ralstonia, were widely distributed in the leachate, indicating the strong adaptive capacity of the microbial population. A correlation analysis between the hydrochemical factors and the microbial community structure revealed that pH was the most influential factor, followed by NO2--N, Fe, Al, Cu, Mn, and others. These results indicate that acidification modifies the hydrochemical conditions of the aquifer, creating an environment that is unfavorable for microbial growth and survival. However, some microorganisms may acquire resistance genes to cope with environmental changes.

Denitrifying bacteria agent together with composite materials enhanced soil chemical properties and denitrifying functions in rare earth tailings: A field study.

The exploitation of ionic rare earth ore using ammonium sulfate extractant in China caused serious soil degradation and nitrogen compounds pollution in surrounding water. It was critical to improve soil properties and eliminate the nitrogen compounds and prevent their diffusion from the rare earth tailings. Here, we addressed this issue by conducting a field experiment for six months through four different treatments including control (CK), denitrifying bacteria agent mainly consisted of Bacillus (DBA), composite materials (CM) and denitrifying bacteria agent together with composite materials (DBA+CM). Besides, the treatments except CK were also amended with basic soil conditioners. DBA+CM could significantly increase soil pH from 5.01 to 6.84 (p ≤ 0.05). Cation exchange capacity in DBA+CM increased from below detection limit to 2.79 cmol+/kg. DBA+CM possessed the highest removal rate of soil NH4+ (95.14 %) and soil NO3- (66.46 %). Compared to CK, DBA+CM significantly increased the absolute abundance of nirS genes and relative abundance of denitrification, nitrate respiration, and nitrite respiration the most (p ≤ 0.05). Denitrification, nitrate respiration and nirS genes were negatively correlated with soil NO3- (p ≤ 0.05). This study demonstrates denitrifying bacteria agent together with composite materials can be a promising approach to control the pollution of nitrogen compounds in ionic rare earth tailings.

A comparative study of methylene blue adsorption and removal mechanisms by calcium carbonate from different sources.

Efficient removal of organic dye pollution from contaminated water is a concern in the absorbent applications. In this study, a green biogenic calcium carbonate (BCC) absorbent was fabricated using Bacillus licheniformis for the removal of methylene blue (MB) from water. This was found to have superior adsorption capacity compared with abiotic calcium carbonate (ACC) and operate within a broad pH range from 3 to 9. MB adsorption on BCC was physical and exothermic. The hydrophobic features, rough nanoporous microstructure, and organic-inorganic mesoporous structure of the BCC may all be responsible for its favorable adsorption mass transfer. The adsorption energy of BCC had a more negative value than that of ACC, indicating a stronger MB interaction with BCC with a lower energy barrier. Hydrogen bonding and electrostatic attraction were involved in the adsorption process. Overall, the findings established a theoretical foundation for the application of BCC in remediation of MB-contaminated water.

Effects of Moss-Dominated Biocrusts on Soil Microbial Community Structure in an Ionic Rare Earth Tailings Area of Southern China.

Moss-dominated biocrusts are widespread in degraded mining ecosystems and play an important role in soil development and ecosystem primary succession. In this work, the soil microbial community structure under moss-dominated biocrusts in ionic rare earth tailings was investigated to reveal the relationship between different types of moss and taxonomy/function of microbiomes. The results showed that microbial community structure was significantly influenced by four moss species (Claopodium rugulosifolium, Orthotrichum courtoisii, Polytrichum formosum, and Taxiphyllum giraldii). The microbial assembly was more prominent in Claopodium rugulosifolium soil than in the other moss soils, which covers 482 bacterial genera (including 130 specific genera) and 338 fungal genera (including 72 specific genera), and the specific genus is 40% to 1300% higher than that of the other three mosses. Although only 141 and 140 operational taxonomic units (OTUs) rooted in bacterial and fungal clusters, respectively, were shared by all four mosses grown in ionic rare earth tailings, this core microbiome could represent a large fraction (28.2% and 38.7%, respectively) of all sequence reads. The bacterial population and representation are the most abundant, which mainly includes Sphingomonas, Clostridium_sensu_stricto_1, and unclassified filamentous bacteria and chloroplasts, while the fungi population is relatively singular. The results also show that biocrust dominated by moss has a positive effect on soil microbe activity and soil nutrient conditions. Overall, these findings emphasize the importance of developing moss-dominated biocrusts as hotspots of ecosystem functioning and precious microbial genetic resources in degraded rare-earth mining areas and promoting a better understanding of biocrust ecology in humid climates under global change scenarios.

Transcriptome expression analysis of the gene regulation mechanism of bacterial mineralization tolerance to high concentrations of Cd2.

Cadmium (Cd) pollution is a pressing environmental issue that must be addressed. In recent years, microbial mineralization biotechnology has been developed into an effective and eco-friendly heavy metal bioremediation solution. In the present research, RNA-Seq technology was utilized to reveal the molecular mechanism through which Bacillus velezensis LB002 induced the mineralization and Cd2+ fixation under high-concentration Cd2+ stress. The metabolic pathways involved in the genes that were significant differentially expressed in the process of bacterial mineralization were also investigated. The results showed that the physiological response of bacteria to Cd2+ toxicity may include bacterial chemotaxis, siderophore complexation, and transport across cell membranes. Bacteria subjected to high-concentration Cd2+ stress can up-regulate genes of argH, argF, hutU, hutH, lpdA, and acnA related to arginine synthesis, histidine metabolism, and citric acid cycle metabolism pathways, inducing vaterite formation and Cd2+ fixation. Thus, the toxicity of Cd2+ was decreased and bacteria were allowed to grow. Real-time quantitative polymerase chain reaction (RT-qPCR) results confirmed the data obtained by RNA-Seq, indicating that bacteria can reduce Cd2+ toxicity by regulating the expression of related genes to induce mineralization. A basic bioremediation strategy to deal with high-concentration heavy-metal pollution was proposed from the perspective of gene regulation.

Effects of composite materials and revegetation on soil nutrients, chemical and microbial properties in rare earth tailings.

The mining of ionic rare earth elements in Ganzhou left large area of barren tailings with severe vegetation destruction in pressing needs of remediation. However, the remediating effects of soil additives combined with revegetation on the preservation of nutrients in the tailings and microbial communities were rarely studied. For this purpose, pilot experiments were implemented in a field, with the control group (CK) only cultivating plants without adding materials, and three treatments including peanut straw biochar composite (T1), phosphorus­magnesium composite (T2) and modified zeolite composite (T3) along with the cultivation of Medicago sativa L., Paspalum vaginatum Sw. and Lolium perenne L. Soil pH and organic matter in CK significantly decreased from 4.90 to 4.17 and from 6.62 g/kg to 3.87 g/kg after six months, respectively (p ≤ 0.05), while all the treatments could effectively buffer soil acidification (over 5.74) and delay the loss of soil organic matter. Soil cation exchange capacity was still below the detection limit in all the groups except T2. The results of rainfall runoff monitoring indicated that compared with CK, only T2 could significantly reduce the runoff loss of soil NO3- and SO42- by 45.61 %-75.78 % and 64.03 %-76.12 %, respectively (p ≤ 0.05). Compared with CK, the bacterial diversity in T2 and T3 significantly increased 21.18 % and 28.15 %, respectively (p ≤ 0.05), while T1 didn't change the bacterial or fungal diversity (p > 0.05). Co-occurrence network analysis showed that compared with CK, the whole microbial communities interacted more closely in the three treatments. Functional prediction of the microbial communities revealed all the treatments were dominated by carbon transforming bacteria and saprotrophic fungi except T2. This study demonstrated that the composite materials combined with revegetation couldn't retain soil nitrogen compounds and sulfate in rare earth tailings in the long term.

Bio-mineralisation, characterization, and stability of calcium carbonate containing organic matter.

The composition of organic matter in biogenic calcium carbonate has long been a mystery, and its role has not received sufficient attention. This study is aimed at elucidating the bio-mineralisation and stability of amorphous calcium carbonate (ACC) and vaterite containing organic matter, as induced by Bacillus subtilis. The results showed that the bacteria could induce various structural forms of CaCO3, such as biogenic ACC (BACC) or biogenic vaterite (BV), using the bacterial cells as their template, and the carbonic anhydrase secreted by the bacteria plays an important role in the mineralisation of CaCO3. The effects of Ca2+ concentration on the crystal structure of CaCO3 were ascertained; when the amount of CaCl2 increased from 0.1% (m/v) to 0.8% (m/v), the ACC was transformed to polycrystalline vaterite. The XRD results demonstrated that the ACC and vaterite have good stability in air or deionised water for one year, or even when heated to 200 °C or 300 °C for 2 h. Moreover, the FTIR results indicated that the BACC or BV is rich in organic matter, and the contents of organic matter in biogenic ACC and vaterite are 39.67 wt% and 28.47 wt%, respectively. The results of bio-mimetic mineralisation experiments suggest that the protein secreted by bacterial metabolism may be inclined to inhibit the formation of calcite, while polysaccharide may be inclined to promote the formation of vaterite. Our findings advance our knowledge of the CaCO3 family and are valuable for future research into organic-CaCO3 complexes.

Non-competitive and competitive adsorption of Cd2+, Ni2+, and Cu2+ by biogenic vaterite.

Ubiquitous bio-minerals exert significant effects on the migration and transformation of metal ions in the environment, however, research into the adsorption of heavy metals by biogenic vaterite (BV) has rarely been reported. The aim of our research was to evaluate the removal effects of Cd2+, Ni2+, and Cu2+ in single and multi-metal ion aqueous solutions using BV induced by Bacillus subtilis. The results demonstrate that the adsorption data of BV for metal ions are more accurately fitted to the Langmuir model compared with the Freundlich model. The max adsorption capacity (mg/g) order of BV was Ni (270.27) > Cu (178.57) > Cd (172.41) in a single-metal system, and Cu (175.44) > Ni (94.34) > Cd (30.30) in a multi-metal system (pH = 5.0, 2.5 g/L). A competitive effect exists amongst heavy metals in multi-metal ion systems, and Cu2+ adsorption is less affected by other two ions. Furthermore, BV can maintain favourable adsorption characteristics even in a very strong acidic environment (pH = 3.0), and its adsorptive capability becomes more favourable at higher temperatures. Kinetic analysis shows that the adsorption process can be better described by a pseudo-second-order model. XRD, FTIR, and SEM-EDS results reveal that metal ion adsorption on BV mostly happened through physical means, and the favourable adsorption characteristics of BV might be attributable to its larger specific surface area, aggregated spherical polyporous and organic-inorganic structure.

Immobilisation of Cd(II) on biogenic and abiotic calcium carbonate.

Ubiquitous calcium carbonate (biogenic or abiotic) exerted an important impact on the migration and transformation of heavy metal in the environment. Cd(II) pollution, common in China, has attracted much attention due to its critical toxicity. The purpose of this study is to compare the differences in adsorption and desorption characteristics of Cd(II) between biogenic CaCO3 (BCa) induced by Bacillus subtilis and abiotic CaCO3 [AR (analytical reagent grade)-CaCO3 and limestone]. The results show that the adsorption data of BCa and abiotic CaCO3 for Cd(II) more closely followed the Langmuir model compared to the Freundlich model. The maximum adsorption capacity (obtained from Langmuir isotherm) of BCa for Cd(II) (172.41 mg/g) was significantly greater than that of abiotic CaCO3 (AR-CaCO3: 6.31 mg/g, limestone: 21.01 mg/g), and its maximum desorption rate (1.48%) was significantly lower than that of abiotic CaCO3 (AR-CaCO3: 74.30%, limestone: 5.65%). Furthermore, the adsorption process of BCa is a spontaneous endothermic reaction and obeys pseudo-second-order kinetics. The obvious advantages of BCa are that it is easily obtained by bacterial culture, and another is its stronger immobilisation of Cd(II) compared to abiotic CaCO3, giving a promising potential for heavy metal remediation.

The Potential Correlation Between Bacterial Sporulation and the Characteristic Flavor of Chinese Maotai Liquor.

The relationship between the formation of characteristic Maotai-flavor substances (MTFS) and the dominant bacteria in Maotai Daqu (MTDQ) has long been a topic of research interest in the field of liquor brewing in China. To investigate the connection between MTFS and the Bacillus subtilis (one of dominant bacteria in MTDQ) cultured on solid plates of wheat extract medium at, temperatures of 37, 46, and up to 55°C (Group A), and at a constant 37°C (Group B), the transcriptomes of the bacteria grown in the two groups were studied. About 10 out of 84 differentially expressed genes (DEGs) were related to promoting sporulation. Furthermore, observations made with transmission electron microscopy (TEM) showed that a thicker spore cortex appeared in Group A. The content of 2, 6-pyridinedicarboxylic acid (DPA), an important component of the spore, was 49.77 (±2.50) and 38.23 (±3.96) µg/mg of dried spores from the bacteria cultured in Groups A and B, respectively. Combined with the production process of Maotai liquor, more DPA accumulates in the high-temperature fermentation stage and is then released by spore germination during the subsequent temperature-drop stage. We suggest that DPA (or its derivatives) can then be transformed into MTFS by the Maillard reaction after many rounds of microbial fermentation. The viewpoint that there is a potential correlation between bacterial sporulation and the production of MTFS is proposed.

Adsorption and Desorption Characteristics of Cd2+ and Pb2+ by Micro and Nano-sized Biogenic CaCO3.

The purpose of this study was to elucidate the characteristics and mechanisms of adsorption and desorption for heavy metals by micro and nano-sized biogenic CaCO3 induced by Bacillus subtilis, and the pH effect on adsorption was investigated. The results showed that the adsorption characteristics of Cd2+ and Pb2+ are well described by the Langmuir adsorption isothermal equation, and the maximum adsorption amounts for Cd2+ and Pb2+ were 94.340 and 416.667 mg/g, respectively. The maximum removal efficiencies were 97% for Cd2+, 100% for Pb2+, and the desorption rate was smaller than 3%. Further experiments revealed that the biogenic CaCO3 could maintain its high adsorption capability for heavy metals within wide pH ranges (3-8). The FTIR and XRD results showed that, after the biogenic CaCO3 adsorbed Cd2+ or Pb2+, it did not produce a new phase, which indicated that biogenic CaCO3 and heavy metal ions were governed by a physical adsorption process, and the high adsorptive capacity of biogenic CaCO3 for Cd2+ and Pb2+ were mainly attributed to its large total specific surface area. The findings could improve the state of knowledge about biogenic CaCO3 formation in the environment and its potential roles in the biogeochemical cycles of heavy metals.

Nitrogen leaching losses following biogas slurry irrigation to purple soil of the Three Gorges Reservoir Area.

Ammonium (NH4+) in biogas slurries is readily nitrified into very mobile soil nitrate (NO3-) to promote nitrogen (N) leaching concerning which a few studies, however, have been reported. These slurries are regularly applied through irrigation to purple soil in the Three Gorges Reservoir Area, and therefore we explored the consequent N leaching there with a plot experiment. Biogas slurry irrigation was carried out with nitrogen application rates of 0, 48, 144, 240, 336, and 480 kg N/ha. As a result, the last two rates have triggered N leaching being detrimental to groundwater safety. In addition, N leaching was negatively correlated with soil microbial biomass, diversity, and respiration, indicating a potential technique to prevent it with soil heterotrophs activated by increased ratios of carbon to nitrogen (C/N) in biogas slurries.

The Red Light Receptor Phytochrome B Directly Enhances Substrate-E3 Ligase Interactions to Attenuate Ethylene Responses.

Plants germinating under subterranean darkness assume skotomorphogenesis, a developmental program strengthened by ethylene in response to mechanical pressure of soil. Upon reaching the surface, light triggers a dramatic developmental transition termed de-etiolation that requires immediate termination of ethylene responses. Here, we report that light activation of photoreceptor phyB results in rapid degradation of EIN3, the master transcription factor in the ethylene signaling pathway. As a result, light rapidly and efficiently represses ethylene actions. Specifically, phyB directly interacts with EIN3 in a light-dependent manner and also physically associates with F box protein EBFs. The light-activated association of phyB, EIN3, and EBF1/EBF2 proteins stimulates robust EIN3 degradation by SCFEBF1/EBF2 E3 ligases. We reveal that phyB manipulates substrate-E3 ligase interactions in a light-dependent manner, thus directly controlling the stability of EIN3. Our findings illustrate a mechanistic model of how plants transduce light information to immediately turn off ethylene signaling for de-etiolation initiation.

Seedlings Transduce the Depth and Mechanical Pressure of Covering Soil Using COP1 and Ethylene to Regulate EBF1/EBF2 for Soil Emergence.

The survival of seed plants in natural environments requires the successful emergence from the soil. In this process, the ethylene signaling pathway is utilized by plants to sense and respond to the mechanical resistance of the soil. Here, we report that constitutive photomorphogenesis 1 (COP1), a central repressor of light signaling, is a key component required for seedlings to sense the depth of soil overlay. Mutation in COP1 causes severe defects in penetrating soil, due to decreased level of EIN3, a master transcription factor in ethylene pathway that mediates seedling emergence. We show that COP1 directly targets the F box proteins EBF1 and EBF2 for ubiquitination and degradation, thus stabilizing EIN3. As seedlings grow toward the surface, the depth of soil overlay decreases, resulting in a gradual increase of light fluences. COP1 channels the light signals, while ethylene transduces the information on soil mechanical conditions, which cooperatively control EIN3 protein levels to promote seedling emergence from the soil. The COP1-EBF1/2-EIN3 module reveals a mechanism by which plants sense the depth to surface and uncovers a novel regulatory paradigm of an ubiquitin E3 ligase cascade.