Ran Duan

Arsenic biotransformation by arsenic-resistant fungi Trichoderma asperellum SM-12F1, Penicillium janthinellum SM-12F4, and Fusarium oxysporum CZ-8F1

Bioremediation of arsenic (As)-contaminated soil using microorganisms has been a focus of research because it is environment friendly and cost-effective. The As-resistant fungi Trichoderma asperellum SM-12F1, Penicillium janthinellum SM-12F4, and Fusarium oxysporum CZ-8F1 were exposed to 50 mg l(-1) of As(V), and the biotransformation of As and the concomitant variance of Eh and pH in the media were studied. Fresh weights of all three isolates increased and then decreased depended on cultivation period. After cultivation for 2 or 3 days, the As(V) added to the media had been completely changed into As(III), whilst As(V) was predominate in fungal cells with concomitantly little As(III) during cultivation. After 15 days, little monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) besides of As(V) and As(III) were found in the cells of T. asperellum SM-12F1, and the total As content was the highest in cells of P. janthinellum SM-12F4 (about 41.5 μg) according to the quantitative analysis of As speciation in cultures. Moreover, when cultivation period reached 3 days, the Eh and pH in the media of T. asperellum SM-12F1 (312.5 mV and 4.8), P. janthinellum SM-12F4 (411.1 mV and 4.2), and F. oxysporum CZ-8F1 (269.4 mV and 4.8) might not responsible for the reduction of As(V) based on the previous study. Therefore, it is speculated that import/export, reduction, and methylation of As are conducted in fungal cells. Future studies investigating the biochemical behaviour of fungi responding to As are needed to gain a better understanding of bioremediation of As-contaminated soils.

Inoculation with chlamydospores of Trichoderma asperellum SM-12F1 accelerated arsenic volatilization and influenced arsenic availability in soils

Abstract Fungi capable of arsenic (As) accumulation and volatilization are hoped to tackle As-contaminated environment in the future. However, little data is available regarding their performances in field soils. In this study, the chlamydospores of Trichoderma asperellum SM-12F1 capable of As resistance, accumulation, and volatilization were inoculated into As-contaminated Chen-zhou (CZ) and Shimen (SM) soils, and subsequently As volatilization and availability were assessed. The results indicated that T. asperellum SM-12F1 could reproduce well in As-contaminated soils. After cultivated for 42 days, the colony forming units (cfu) of T. asperellum SM-12F1 in CZ and SM soils reached 10 10 −10 11 cfu g −1 fresh soil when inoculated at a rate of 5.0%. Inoculation with chlamydospores of T. asperellum SM-12F1 could significantly accelerate As volatilization from soils. The contents of volatilized As from CZ and SM soils after being inoculated with chlamydospores at a rate of 5.0% for 42 days were 2.0 and 0.6 μg kg −1 , respectively, which were about 27.5 and 2.5 times higher than their corresponding controls of no inoculation (CZ, 0.1 μg kg −1 ; SM, 0.3 μg kg −1 ). Furthermore, the available As content in SM soils was decreased by 23.7%, and that in CZ soils increased by 3.3% compared with their corresponding controls. Further studies showed that soil pH values significantly decreased as a function of cultivation time or the inoculation level of chlamydospores. The pH values in CZ and SM soils after being inoculated with 5.0% of chlamydospores for 42 days were 6.04 and 6.02, respectively, which were lowered by 0.34 and 1.21 compared with their corresponding controls (CZ, 6.38; SM, 7.23). The changes in soil pH and As-binding fractions after inoculation might be responsible for the changes in As availability. These observations could shed light on the future remediation of As-contaminated soils using fungi.

Arsenic speciation transformation and arsenite influx and efflux across the cell membrane of fungi investigated using HPLC-HG-AFS and in-situ XANES.

Microorganisms dominated speciation of arsenic (As) play an important role in the biogeochemical cycling of As. In the study, species transformation of arsenite [As(III)] and As(III) influx and efflux across the cell membranes of Trichoderma asperellum SM-12F1, Penicillium janthinellum SM-12F4, and Fusarium oxysporum CZ-8F1 cells were studied using a cellular lysis plus chromatographic separation method and further the in-situ X-ray absorption near edge structure (XANES) analysis. The results indicated that As(III) can enter into fungal cells and that a portion of the As(III) can be exuded out of cells. For both As sequestrated into fungal cytoplasm and As adsorbtion onto cell walls, As(III) was found to be the dominated form of As. XANES analysis showed that As(III) accounted for 58.4%, 59.5%, and 73.0% of the total As in the cells of T. asperellum SM-12F1, P. janthinellum SM-12F4, and F. oxysporum CZ-8F1, respectively. Among these fungal strains, however, there were obvious differences in the relative proportions of arsenate [As(V)], monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA). For T. asperellum SM-12F1, the proportion (%) of MMA was 31.1%, and no As(V) or DMA was detected. For F. oxysporum CZ-8F1, the proportions of As(V) and MMA were 15.8% and 8.8%, respectively, but no DMA was observed. As(V), MMA, and DMA accounted for 4.2%, 29.5%, and 8.1%, respectively, of the P. janthinellum SM-12F4 cells. Some of the intracellular As(III) can be oxidated and methylated by these fungal strains and yield As(V), MMA, and DMA as products.

Demethylation of arsenic limits its volatilization in fungi.

Arsenic (As) biomethylation is increasingly being regarded as a promising method to volatize As from the environment; however, the As volatilization efficiency of most microorganisms is low. Here, the speciation transformation of dimethylarsinic acid (DMA) as an important methylation intermediate in the cells of Fusarium oxysporum CZ-8F1, Penicillium janthinellum SM-12F4, and Trichoderma asperellum SM-12F1 were investigated. These fungal strains have been certified to volatilize As from As-loaded environment. In situ X-ray absorption near edge structure (XANES) indicated that demethylation of DMA with methylarsonic acid (MMA), arsenate [As(V)], and arsenite [As(III)] as intermediates or products occurred in fungal cells after exposure to DMA for 15 days. 36.7-55.7% of the original DMA could lose one or two methyl groups and be changed into MMA or inorganic As. Chromatographic separation of the cell lysates also supported these findings. Thus it comes that demethylation might be a remarkable internal factor limiting As volatilization efficiency.

Effects of different fertilizer application methods on the community of nitrifiers and denitrifiers in a paddy soil

PurposeNitrifiers and denitrifiers are the key drivers of N cycling in paddy soil. Little is known about the effects of different fertilization methods, especially side bar fertilization, on the community of nitrifiers and denitrifiers in paddy soils. Here we assess the relationships between soil physicochemical properties, denitrification and nitrification activities, and the underlying microbial communities in a surface layer of paddy field soil treated with different fertilization methods.Materials and methodsSoil was unfertilized (control), treated with conventional chemical fertilizer (CF), CF plus pig manure (MC), or slow-release fertilizer (SR), or by slow-release side bar fertilization (SB). Soil was sampled after one season of early and late rice growth. We determined soil physicochemical properties, potential nitrification rates (PNR), and denitrification enzyme activities (DEA). Ammonia-oxidizing archaeal (AOA) and bacterial (AOB) communities were assessed via their ammonia monooxygenase (amoA) genes, and denitrifiers via nitrite reductases (nirK and nirS) and nitrous oxide reductase (nosZ). Quantitative PCR was used to assess gene abundance, terminal restriction fragment polymorphism (T-RFLP) to investigate fertilization effects on microbial communities, and clone library sequencing and phylogenetic analysis to explore the taxonomic diversity of the nitrifiers and denitrifiers.ResultsFertilization significantly increased the amount of NH4+-N in the soil of SB and MC treatments, whereas MC lowered the NO3−-N level. DEA was higher for MC and CF than the other treatments. The PNR in MC-treated soil was significantly lower than that in CF-treated soil. There were no significant differences in AOA and nirS copy numbers; however, nirK and nosZ copy numbers were higher for MC compared with CF. The number of AOA terminal restriction fragments (TRFs) increased significantly with N addition and reached the highest level for SB, whereas the number of AOB TRFs did not change significantly between treatments. Similarly, the number of nirK TRFs increased under fertilization, with the highest number obtained for SR; however, no significant change was observed for nirS and nosZ TRFs across different treatments, except for their relative abundance. All AOA amoA genotypes were in archaeal group 1.1b, whereas 95% AOB were in Nitrosospira cluster 3a. More than 40% of nirS OTUs were affiliated with Herbaspirillum, a key N-cycle player in this paddy soil.ConclusionsThe SB and MC treatments had significant effects on soil N, DEA, and PNR levels, and affected the community of N-functional microbes. SB in combination with pig manure would contribute to the improvement of paddy soil fertility.

Reduced arsenic availability and plant uptake and improved soil microbial diversity through combined addition of ferrihydrite and Trichoderma asperellum SM-12F1

Arsenic (As) accumulation in agricultural soils is prone to crop uptake, posing risk to human health. Passivation shows potential to inactivate soil labile As and lower crop As uptake but often contributes little to improving the microbiota in As-contaminated soils. Here, the combined addition of ferrihydrite and Trichoderma asperellum SM-12F1 as a potential future application for remediation of As-contaminated soil was studied via pot experiments. The results indicated that, compared with the control treatment, the combined addition of ferrihydrite and T. asperellum SM-12F1 significantly increased water spinach shoot and root biomass by 134 and 138%, respectively, and lowered As content in shoot and root by 37 and 34%, respectively. Soil available As decreased by 40% after the combined addition. The variances in soil pH and As fractionation and speciation were responsible for the changes in soil As availability. Importantly, the combined addition greatly increased the total phospholipid fatty acids (PLFAs) and gram-positive (G+), gram-negative (G−), actinobacterial, bacterial, fungal PLFAs by 114, 68, 276, 292, 133, and 626%, respectively, compared with the control treatment. Correspondingly, the soil enzyme activities closely associated with carbon, nitrogen, and phosphorus mineralization and antioxidant activity were improved. The combination of ferrihydrite and T. asperellum SM-12F1 in soils did not reduce their independent effects.