| The effects of Aluminum on soil microbial communities, soil microbial biomass carbon (Cmic) and soil enzymes were studied by using red acidic soil(forest and agricultural soils) with a short-term treatment in this thesis. Biological community assessments included culturable bacteria (traditional techniques), soil enzyme activities, changes in microbial (bacteria, fungi and achaea) community structures (PCR-amplified Restriction Fragment Length Polymorphism (RFLP) analysis). Moreover, a Al-tolerance bacterium (SB1) and two Al-tolerance fungi (S4 and S7) were isolated and identified, and their tolerance characteristics were investigated. The results will be provided valuable bio-materials for the remediation of acidic red soil which contains high level of monomeric Al. Also, to some extent, this study may provide some scientific basis for the evaluation on the potential risks of the increasing level of Al3+ due to the serious acid deposition in the soil-water system. The main results are reported as follows:1. In the present study, under the stress of available Al of Chingkang mountains red soil, such effects were measured by the changes of microbial numbers, enzyme activities (URE and ACP) and microbial biomass carbon (Cmic). The results showed that the microbial numbers, Cmic, ACP and URE activities in the Al-treated samples were significantly lower (p<0.05) than those in control, and clear differences between agricultural soil (AGR soil) and forest soil (FOR soil) were found. The average number of bacteria and fungi was significantly inhibited with an addition of Al3+ higher than 80 mg kg"1 (for bacteria) and 160 mg kg-1 (for fungi), and the decrease in the number of fungus was more prominent in AGR soil than in FOR soil. URE activities decreased significantly only in the early incubation days and were obviously suppressed at the higher level of Al3+(240 mg kg-1). AGR soil had a lower effect on pH and ACP activities in the case of Al3+ additions than FOR soil. Moreover, a negative correlation was found between monomeric Al concentration and microbial numbers, enzyme activities and soil Cmic.2. The soil bacterial communities in response to Al treatments were evaluated by PCR-amplified restriction fragment length polymorphism (PCR-RFLP) of 16S rRNA genes in acidic red soil samples in this study. A total of 6 bacterial communities were sampled from two representative soil types under different Al-treated concentrations. Bacterial genomic DNA was extracted and nested PCR-amplified to obtain 16S rDNA fragments which were cloned to construct 6 16S rDNA libraries. Clones of each library were selected randomly for PCR-RFLP analysis of rDNA fragments, and eventually 60 genotypes were identified by RFLP fingerprintings. These 60 genotypes were sequenced and their respective phylotype was identified through the Blast tool of NCBI (similarity 93-100%) and phylogenetic analyzed. The phylotype richness, frequency distribution (evenness), and composition of the clone libraries were investigated by using a variety of diversity indices. Among these phylotypes,96.7% belonged to the genera ?-,?-, ?-,?- Proteobacteria, Acidobacteria, Cytophaga-Flavobacteria-Bacteroides (CFB group), Green Nonsulfur bacteria (GNS), Gemmatimonadetes, high GC Gram" andNitrospira. Sequence analyses revealed that 56.7%(34) of clone sequences were similar to those of uncultured soil bacteria in the environment. In addition, the bacterial diversities and compositions clearly displayed in different soil samples. More genera were discovered in Ao soil sample than any others, and some special species, such as Nitrospira, disappeared in Al-treated soils. 3. To analysis the fungal community composition properties of acidic red soil in typical forest and agricultural soils under different aluminum stress, we constructed fungus ITS rDNAs library construction using PCR-RFLP technology (including direct amplification, cloning, enzyme digestion and sequence analysis). Clones of each library were selected randomly for PCR-RFLP analysis of rDNA fragments with Rsa I and Hal I digestion, and eventually 77 genotypes were identified from 6 ITS rDNA libraries by RFLP fingerprintings. These 77 genotypes were sequenced and their respective phylotype was identified through the Blast tool of NCBI (similarity 95-100%) and phylogenetic analyzed. The phylotype richness, frequency distribution (evenness), and composition of the clone libraries were investigated by using a variety of diversity indices. Among these phylotypes,79.2%(61) belonged to Ascomycota, Basidiomycota and Glomeromycota. Sequence analyses revealed that 20.8%(16) of clone sequences were similar to those of uncultured soil fungi in the environment. Members of the Basidiomycota were the dominant groups in both FOR and AGR soils, accounting for close to1/2 of the total population of soil fungi, but the species are less than Ascomycota. In additional, the abundance of Basidiomycota is significant decreased in two soils as the levels of Al3+ increased. At the same time, the abundance of Ascomycota is significant increased and becomes the dominant population in F2 and A2. So, Al3+ added can affect fungal population quantity and distribution.4. The abundance and diversity of soil Archaea in response to Al treatments were evaluated by PCR-amplified restriction fragment length polymorphism (PCR-RPLP) of 16S rRNA genes in acidic red soil samples in this study. Rarefaction curves for acidic red soil samples OTUs of 16s rDNAs gene clones in different Al treatment draw by Estimate S software showed that 16S rDNA libraries could reflect the Archaea community diversities in all soil samples. There are three types of Archaea in the acidic red soils:Crenarchaeota, Euryarchaeota and unclassified Archaea. Among them, Crenarchaeota accounted for the highest proportion. We compared the distribution of Archaea communities among the 6 soil samples using Mothur. The results show that the unclassified Archaea accounted for the highest proportion in Ao and Fo, suggesting that the special environment in the acidic soil may influence special Archaea type composition. Moreover, the abundance of Crenarchaeota is significant decreased in two soils as the levels of Al3+ increased, meanning that Crenarchaeota owned higher Al-tolerance and gradually evolved as the dominant population at high concentrations of aluminum.5. We isolated 8 strains of bacteria which had stronger ability of aluminum resistant (reach 5 mmol/L) from acidic red soils through the traditional selective enrichment culture method. PCR-RFLP results showed that these 8 strains showed the same genotype. The genotype was sequenced and identified through the Blast tool of NCBI. The phylotype belonged to ?-proteobacteria (similarity 100%). The sequence submitted to the DDBJ and set "Burkholderia sp. SB1" as the strain name. This was the first report on Burkholderia sp. which had strong aluminum tolerance in acidic red soils.6. We studied the tolerance of bacterial SB1on acid, temperature, antibiotic under the experimental simulation condition, and studied the morphological characteristics of SB1 under different aluminum concentrations using SEM. Furthermore, we studied the adsorption properties of aluminum through the ICP-MS. These results showed that aluminum stress prolonged SB 1 into the exponential growth phase time, but had little effect on the bacterial growth period. SB1 with strong aluminum tolerance can grow in pH 2.2 culture medium when aluminum did not exist. The presence of aluminum can increase the acid on bacterial growth toxicity. The optimum growth temperature of SB1 is about 37?. Appropriate to reduce the temperature has little effect on bacterial growth, but high temperature has a significant effect on bacterial growth. SB1 can absorb a certain amount of aluminum and saturatesoon at high Al concentrations. In additional, SB1 can change their shape structure to adapt to high Al stress environment.7. We isolated 2 strains of fungi which had stronger ability of aluminum resistant (reach 550 mmol/L) from acidic red soils through the traditional selective enrichment culture method.26S rRNA identification and RDP classifier results showed that S4 and S7 belonged to Trichocomaceae (similarity 95-100%), and S4 belonged to Eupenicillium, S7 belonged to unclassified Trichocomaceae.8. Aluminum induced pH decrease is not the main reason of biological growth inhibition. Fungi S4 and S7 had a very strong aluminum tolerance. Our results showed strains S4 and S7 had strong aluminum adsorption capacity, and S4 had stronger adsorption capacity than S7. Even so, S7 show more powerful aluminum tolerance than S4, mainly through the secretion of more extracellular substances chelated aluminum to achieve. |
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