| The mineral-microbe interactions lie at the heart of geomicrobiological processes.Minerals not only can serve as physical supports for microbial attachment,but also provide nutrients and electron donor or receptor essential to microbial growth.The physical attributes and chemical compositions of minerals can influence the structure of microbial communities.Meanwhile microbial activities can also impact mineral weathering and formation through different mechanisms.Microbe-mediated weathering of mineral phosphates(known as phosphate-solubilizing microorganisms,PSMs)is a research hotspot of mineral-microbe interactions.Given that phosphorus(P)is an essential macronutrients required for living organisms and the preferred form of P for organism growth is dissolved inorganic orthophosphate(PO43-),PSMs play a key role in sustaining the stability of ecosystems through mobilization of P into bioavailable form.Isolation and identification of natural PSMs have been conducted from surface soil environments.However,little is known about PSMs in subsurface ecosystem.Karst cave is formed in the soluble rocks through water-rock interaction and can serve as natural laboratories to study such subterranean microbial diversity and geomicrobiological processes.In this study,we have explored the bacterial and fungal diversity in the weathered rocks of Heshang Cave,a typical karst cave,central China.Particular attention was given to the impact of mineral compositions on the bacterial and fungal community structures.Furthermore,we conduct a comprehensive investigation on PSMs in the subsurface ecosystem.Our main results are listed below.1)The weathered rock wall samples of Heshang cave can be divided into surface,crust lamination and loose lamination according to the color and hardness.TOC content varied among different laminations and was lower in crust laminations.The p H was near neutral and similar among different laminatios.Minerals of rock samples collected from photic zone mainly consist of carbonate minerals(including dolomite,calcite,Mg-calcite and aragonite)and quartz.But in twilight and aphotic zones phosphate minerals such as apatite(hydroxyl-,fluor-)and whitlockite were frequently detected besides carbonate minerals.Other minerals such as illite-2M and gypsum also presented in twilight and aphotic zone occasionally.DCA analysis result also showed that signigicant difference exsits in mineral compositions among different sampling zones,also among different laminations.2)A total of 32 bacterial phyla were identified from cave weathered rocks and the phylum Actinobacteria(accounting for 52.14%)was the most abundant bacterial group in most rock samples.Apart from Actinobacteria,phylum Chloroflexi(10.95%),Planctomycetes(10.29%)and Proteobacteria(8.85%)also occurred in rock samples with a high relative abundance.At the genus level,only 17.33% of the sequences could be affiliated with specific genera for bacterial communitie,and six phosphate solibilizing bacterial genus including Streptomyces,Bacillus,Pseudomonas,Agrobacterium,Serratia and Flavobacterium could been found in the weathered rocks with a very low abundance,which the total abundance was just 0.4%.The ACE estimators showed that the richness of bacterial communities ranged from 1482 to 2692 taxa among different samples.The range of the Shannon diversity index for the bacterial communities was 5.83 to 9.01.Analysis of variance results showed that neither ACE estimators nor Shannon indices had significantly variation in terms of spatial or laminational.NMDS biplot based on weighted unifrac distance showed that photic zone had a distinct bacterial communities with twilight and aphotic zone.Bacterial communities varied slightly among three weathered laminations within the same sampling zone RDA analyses showed that the TOC content(p=0.006)and phosphate minerals(p=0.022)affected bacterial communities significantly.3)Only 5 fungal phyla(Ascomycota,Basidiomycota,Glomeromycota,Rozellomycota and Zygomycota)were identified in weathered rock samples,phylum Ascomycota(accounting for 91.03%)covered most fungal community.Within the phylum Ascomycota,class Sordariomycetes(45.89%)take the most part,class Eurotiomycetes(9.50%)and Dothideomycetes(8.90%)were also relative abundant in weathered rocks.There were more sequences(40.92%)that could be affiliated with specific genera and three phsophate solibilizing fungal genus including Penicillium,Aspergillus and Fusarium could been found in the rock sampls with relative abundance were 7.00%,1.00% and 0.33%,respectively.Moreover,the relative abundance of three phosphate solubilzing fungal genus were much higher in photic zone.NMDS biplot of fungal communities also showed that fungal comunities in photic zone had a distinct bacterial communities with twilight and aphotic zone but less scales than bacterial communities,no distinct clusters could been observed in the fungal communities among different laminations within each zone.RDA analyses showed that only phosphate minerals(p=0.012)affected fungal communities significantly.4)The detecting of mineral phosphate-solubilizing function in PVK medium showed that a better MPS trait could been obeserved in the rock samples collected from photic and twilight zone then aphotic zone.Both solid and liquit NBRIP medium containing 5 g of tricalcium phosphate(TCP)was used for measuring the microbial mineral phosphate-solubilizing trait after isolated and purely cultured by R2 A and PDA mediun for rocks bacterial and fungal strains.In present work,we have detected 39 bacterial and 28 fungal isolates among all three sampling zones.After 7days incubation,only one bacterial isolates(HSD-Bi10)produced clear haloes on solid medium,whereas 10 fungal isolates(HSD-F1~3,HSD-F10,HSD-F12~13,HSD-16-17,HSD-F21~22)could produce clear haloes on solid medium.The aqueous orthophosphate-P(Pi)content in all bacterial cultures were ranged from 2.21~394 mg/L and only five bacterial cultuers(HSD-Bi6,HSD-Bi10,HSD-Bi25,HSD-Bi28,HSD-Bi31)surpass 20 mg/L,while the aqueous orthophosphate-P content in non-inoculate control was 3.47 mg/L.The aqueous orthophosphate-P content in fungal cultures ranged from 3.17~399 mg/L and 10 fungal cultures(HSD-F3,HSD-F6,HSD-F10~13,HSD-F16~17,HSD-F21~22 and HSD-F26)surpassed 20 mg/L 。Phosphate solubilizing activity of these strains were associated with a drop in the p H in almost cultures.Identified as by means of morphological observation and 16 S or ITS ribosome gene sequence analysis showed that these phosphate solubilizing bacterial strains were belonged to the gunus Burkholderia,Pseudomonas,Bacillus and Streptomyces respectively,whereas the phosphate solubilizing fungal strains mainly were Aspergillus nigur and Penicillium spp..From the present study,we demonstrate that the weathered rocks in Heshang cave harbor a high diversity of bacterial and fungal communities and show significant spatial differences and vary slighly in different laminations.Actinobacteria and Ascomycetes dominate bacterial and fungal communities in weathered rocks,respectively.High abundance of unknown and uncultured bacterial sequences identified in rock samples which suggests very unique bacterial communities in Heshang Cave,could promote the identification of novel strains.Mineral composition mainly phosphate minerals significantly impacts bacterial and fungal communities.Also,weathered rocks in Hesghang cave habour a diverse group of phosphate solubilizing microbes according both Ilumina sequence and cultures detecting.P-solubilizing activity of these strains mostly are associated with a drop in the p H of the medium. |
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