Soil Bacterial DNA Research Articles

Biodegradability of bacterial cellulose polymer below the soil and its effects on soil bacteria diversity

The increasing demand for sustainable materials has led to a surge in research on biodegradable polymers. This study evaluated the weight and visual changes of bacterial cellulose (BC) polymers and nylon membranes during an eight-week biodegradation study below soil. Scanning electron microscope (SEM) images were taken to investigate morphological changes from decomposition. Changes in soil microbial communities and the relationship between microbial abundance and BC biodegradation were investigated. BC was produced from the fermentation of coffee kombucha using 15 % d-dextrose C6H12O6 (DE) as the nutrient carbohydrate substrate. BCs were purified with 0.5 mol/L aqueous NaOH solution and 1.5 % (v/v) aqueous NaOCl and then freeze-dried. The biodegradation study was conducted using seedling pots and nylon membrane filters inside a greenhouse over eight weeks, with sample points taken at various intervals. Soil characteristics such as pH, nutrient content, texture, organic matter, and soil moisture were analyzed. Results showed that BC polymers underwent significant weight losses and visual decomposition, while nylon membranes did not show any weight or visual losses. Approximately 75 % of the BC polymers were decomposed by the end of the study. Fatty acid methyl ester (FAMEs) analysis showed increased microbial absolute abundance (nmol/g) for BC treatments over biodegradation time. FAMEs principal component analysis (PCA) ordination plots showed the relative abundance of fungi increased in BC microbial communities suggesting fungi's attraction to cellulose. Soil bacterial DNA analysis revealed the dominance of Proteobacteria for all treatments. PCA plots and Faith-pd index Alpha Diversity results showed significant bacterial community variations for week 2 of the biodegradation study compared to the other weeks. Adonis microbial community Beta Diversity analysis showed significant differences between treatments (p = 0.001). PCA plots also showed a slight differentiation of the bacterial communities with BC compared to bacterial communities with nylon and soil (control). This study confirms that BC is easily decomposed in soil and that its biodegradability is likely driven by fungal communities.

The Local Ecological Memory of Soil: Majority and Minority Components of Bacterial Communities in Prehistorical Urns from Schöps (Germany)

Soil samples from five iron-age urns and five outside reference samples are investigated by 16S RNA sequencing (ngs) to evaluate the effect of the urns on the soil bacterial diversity. The urns are approximately 2000 years old and had been found during an archaeological excavation of the wet subground of an agricultural used area. All ten samples show similar distribution of phyla. Some major components show differences between the inside-urn and the outside-urn samples. Thus, the interior of urns is marked by a slightly enhanced percentage of Actinobacteria in comparison with the reference samples, whereas no difference between inside-urn samples and reference samples were found in the Chloroflexi content. Between the minor components, some operational taxonomic units (OTUs) could be proved which were found in several urns, but in no reference samples, among them the group TSCOR001-H18 (bacilli), Fodinicola and Angustibacter, NS11-12 marine group and the group S15-21. In addition, several OTUs were detected in single urns, which had been not provable in all other samples. The comparison between the individual urns and between urns and reference samples suggest that the minor components can be regarded as reflecting a signature-like sample-specific soil bacterial DNA pattern, which might become interesting in future investigations on the comparison of archaeological excavation sites and for the registration of bacterial diversity and gene resources at these places.

Mitigating temporal mismatches in forensic soil microbial profiles

ABSTRACTForensic implementation of soil bacterial DNA profiling is limited by the potential for temporal mismatch of DNA profiles, e.g. after storage or seasonal changes. We compared profiles of samples retrieved at one location over 14 years after air-drying, freeze-drying and –80 °C freezing storage. Sample mismatch in freeze-dried and air-dried samples was significant after two years and continued to increase yearly, whereas profiles after –80 °C freezing remained unchanged for many years. In an attempt to mitigate inter-seasonal temporal mismatches, e.g. when months pass between crime and seizure of evidence, soils sampled in winter and summer were exposed to artificial ‘summer’ and ‘winter’ conditions, respectively, and their DNA profiles were compared. Differences were small between soil types, larger between seasons and largest between ‘natural’ and ‘artificial’ seasons. Understanding sources of temporal variations is critical for storage of forensics samples and for developing mitigation procedures that could help overcome these time-induced limitations.

Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production

Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production

Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production

The aim of this study was to isolate a novel amylomaltase gene from community DNA of soil samples collected from Ban Nong Khrok hot spring in Thailand without bacterial cultivation. Using PCR, a 1.5 kb full-length gene was amplified and ligated with pGEM-T easy vector to transform into Escherichia coli DH5 alpha for sequencing. The obtained gene encoding an amylomaltase consisted of 1.503 bp that translated into 500 amino acids. Amino acid sequence deduced from this gene was highly homologous with that of amylomaltase from Thermus thermophillus ATCC 33923. In order to express the enzyme, the cloned gene was subcloned into plasmid pET-17b and introduced into E. coli BL21 (DE3). The maximum expression was observed when the cloned cells were cultured at 37 degrees C for 6 h with 0.5 mM IPTG induction. By 10% SDS-PAGE, the relative molecular mass of the purified amylomaltase was approximately 58 kDa. This enzyme was optimally active at 70 degrees C and pH 9.0. In addition, the enzyme could hydrolyze pea starch to yield the large-ring cyclodextrins with degrees of polymerization of 23 and higher. It is noted that CD29 was the product in the largest quantity under all tested conditions.

Effects of replacement control with four forage species on bacterial diversity of soil invaded by <I>Flaveria bidentis</I>

Aims Invasive plants can alter soil physicochemical properties by changing the soil microbial community (which is closely related to plant growth and development) and thus further promote the invasion process. Our objectives were to (a) compare changes in soil bacterial diversity with cultivation of four replacement plants (Sorghum bicolor × S. sudanense, Helianthus annuus, Medicago sativa and Lolium perenne) mixed with Flaveria bidentis in different growth period and (b) determine the responses of the soil bacterial community to F. bidentis invasion and replacement. Methods Total soil bacterial DNA was extracted by PowerSoil DNA Isolation Kit, 16S rRNA V3 fragments were amplified with bacterial universal primers, and purified fragments were cloned into pGEM-T-Easy vector. Sequence results were aligned on NCBI. Important findings Soil bacterial diversity was decreased in F. bidentis monoculture and was lower than in the soils of the monocultures of the four replacement plants and in the soils of the replacement plants and F. bidentis. There were Hsignificant differencesH in soil bacterial 16S rRNA denaturing gradient gel electrophoresis (DGGE) fingerprinting patterns between the mixedand monoculture soils. Also, there were characteristic bacterial communities with each mixed culture soil on different dates. The Shannon diversity index of soil bacteria peaked in July and decreased starting in August, and this change was synchronous with plant growth. Soil bacterial diversity was reduced following the invasion of F. bidentis, and that the diversity level was increased by cultivating replacement plants with F. bidentis. Therefore, we proposed that soil bacteria play an important role in F. bidentis invasion and control.

Soil Microbial Community Response to Drought and Precipitation Variability in the Chihuahuan Desert

Increases in the magnitude and variability of precipitation events have been predicted for the Chihuahuan Desert region of West Texas. As patterns of moisture inputs and amounts change, soil microbial communities will respond to these alterations in soil moisture windows. In this study, we examined the soil microbial community structure within three vegetation zones along the Pine Canyon Watershed, an elevation and vegetation gradient in Big Bend National Park, Chihuahuan Desert. Soil samples at each site were obtained in mid-winter (January) and in mid-summer (August) for 2 years to capture a component of the variability in soil temperature and moisture that can occur seasonally and between years along this watershed. Precipitation patterns and amounts differed substantially between years with a drought characterizing most of the second year. Soils were collected during the drought period and following a large rainfall event and compared to soil samples collected during a relatively average season. Structural changes within microbial community in response to site, season, and precipitation patterns were evaluated using fatty acid methyl ester (FAME) and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses. Fungal FAME amounts differed significantly across seasons and sites and greatly outweighed the quantity of bacterial and actinomycete FAME levels for all sites and seasons. The highest fungal FAME levels were obtained in the low desert scrub site and not from the high elevation oak-pine forests. Total bacterial and actinomycete FAME levels did not differ significantly across season and year within any of the three locations along the watershed. Total bacterial and actinomycete FAME levels in the low elevation desert-shrub and grassland sites were slightly higher in the winter than in the summer. Microbial community structure at the high elevation oak-pine forest site was strongly correlated with levels of NH4+-N, % soil moisture, and amounts of soil organic matter irrespective of season. Microbial community structure at the low elevation desert scrub and sotol grasslands sites was most strongly related to soil pH with bacterial and actinobacterial FAME levels accounting for site differences along the gradient. DGGE band counts of amplified soil bacterial DNA were found to differ significantly across sites and season with the highest band counts found in the mid-elevation grassland site. The least number of bands was observed in the high elevation oak-pine forest following the large summer-rain event that occurred after a prolonged drought. Microbial responses to changes in precipitation frequency and amount due to climate change will differ among vegetation zones along this Chihuahuan Desert watershed gradient. Soil bacterial communities at the mid-elevation grasslands site are the most vulnerable to changes in precipitation frequency and timing, while fungal community structure is most vulnerable in the low desert scrub site. The differential susceptibility of the microbial communities to changes in precipitation amounts along the elevation gradient reflects the interactive effects of the soil moisture window duration following a precipitation event and differences in soil heat loads. Amounts and types of carbon inputs may not be as important in regulating microbial structure among vegetation zones within in an arid environment as is the seasonal pattern of soil moisture and the soil heat load profile that characterizes the location.

Response of bacterial community during bioremediation of an oil-polluted soil.

To study the response of the bacterial community to bioremediation of a soil with an aged contamination of crude oil. The bacterial community in laboratory soil columns during a 72-day biostimulation treatment was followed by analysing the number of total cultivable hydrocarbon-degrading bacteria, soil respiratory activity and the 16S-23S rDNA internal transcribed spacer homoduplex heteroduplex polymorphisms (ITS-HHP) of total soil bacterial DNA. ITS-HHP permits an estimate of both length and sequence polymorphism in a 16S-23S rDNA spacer population, using to advantage the homoduplex and heteroduplex fragments that are generated during PCR. The treatment, made by air sparging and biostimulation with a mineral nutrient and surfactant solution, resulted in a 39.5% decrease of the total hydrocarbon content. Within 4 days of treatment onset the bacterial community underwent a first phase of activation that led to a substantial increase in the observable diversity. Subsequently, after a 12-day period of stability, another activation phase was observed with further shifts of the community structure and an increase in the abundance and diversity of catechol-2,3-dioxygenase (C23O) genes. The overall data suggest an important contribution of uncultivable bacteria to the soil bioremediation, since, during the second activation phase, the increases of the respiratory activity, bacterial diversity and C23O gene abundance and diversity were not accompanied by a corresponding increase of the cultivable bacteria number. This study shows that successive phases of activation of bacterial populations occur during a bioremediation treatment of oil-polluted soil.

Diversity amongst Bacillus merA genes amplified from mercury resistant isolates and directly from mercury polluted soil

Mercury resistant (Hg R) Bacillus were isolated from soil at a mercury contaminated site on the banks of the River Mersey, in Northwest England. The frequency of Hg R bacteria in the culturable Bacillus population in soil samples ranged from 4.4% to 5.4%. No Hg R Bacillus could be isolated from a sediment sample taken close to the contaminated soil sample in 1994. DNA sequences homologous to a merA probe from the Hg R isolate Bacillus sp. strain RC607 were found to be chromosomally located in 98% of the Hg R Bacillus isolates from the soil site. Oligonucleotide primers designed to the merA gene of RC607 were used to amplify the sequences present in the isolates, and also merA determinants present in bacterial DNA directly extracted from soil. Classification of cultured Bacillus merA products and of 40 merA determinants amplified directly from extracted soil bacterial DNA was based on restriction fragment length polymorphism patterns. This technique revealed a total of 22 classes of amplification products. Unweighted paired group mean analysis was used to examine the relationships between these classes and indicated the presence of microsites in Fiddlers Ferry soil containing distinct Hg R populations. API identification of the Hg R Bacillus isolates revealed that diversity of merA between these microsites is not simply due to the divergent evolution of differing Bacillus species. Lack of a more substantial class correlation between cultured samples taken at different times indicated significant temporal variation in the genetic composition of Bacillus merA in soil.

Diversity amongst Bacillus merA genes amplified from mercury resistant isolates and directly from mercury polluted soil

Mercury resistant (HgR) Bacillus were isolated from soil at a mercury contaminated site on the banks of the River Mersey, in Northwest England. The frequency of HgR bacteria in the culturable Bacillus population in soil samples ranged from 4.4% to 5.4%. No HgRBacillus could be isolated from a sediment sample taken close to the contaminated soil sample in 1994. DNA sequences homologous to a merA probe from the HgR isolate Bacillus sp. strain RC607 were found to be chromosomally located in 98% of the HgRBacillus isolates from the soil site. Oligonucleotide primers designed to the merA gene of RC607 were used to amplify the sequences present in the isolates, and also merA determinants present in bacterial DNA directly extracted from soil. Classification of cultured Bacillus merA products and of 40 merA determinants amplified directly from extracted soil bacterial DNA was based on restriction fragment length polymorphism patterns. This technique revealed a total of 22 classes of amplification products. Unweighted paired group mean analysis was used to examine the relationships between these classes and indicated the presence of microsites in Fiddlers Ferry soil containing distinct HgR populations. API identification of the HgRBacillus isolates revealed that diversity of merA between these microsites is not simply due to the divergent evolution of differing Bacillus species. Lack of a more substantial class correlation between cultured samples taken at different times indicated significant temporal variation in the genetic composition of Bacillus merA in soil.

DNA Probe Analysis of Microbial Community Selected in Field by Long‐Term 2,4‐D Application

AbstractThe 2,4‐D (2,4‐dichlorophenoxyacetic acid)‐degrading microbial populations selected by 2,4‐D amendments to soil were analyzed by DNA probes during a 4‐yr field study. Two were control subplots not treated with 2,4‐D, and the other six were treated with 2,4‐D in replicates at three different concentrations. Total soil bacterial DNA was regularly extracted from each subplot soil and analyzed on slot and Southern blots using the gene probes tfdA and Spa that detect two different 2,4‐D‐degrading populations. Slot blot analyses revealed that the hybridization signal of the probes to total soil bacterial DNA substantially increased with time and continued 2,4‐D application, indicating a shift from a more diverse 2,4‐D‐degrading microflora to populations that shared greater homology to the two probes. More hybridization signal was obtained with the Spa probe, suggesting that microorganisms with this gene sequence were more dominant than degraders with tfdA‐like genes. The Southern blot band patterns indicated that the dominant Spa‐hybridizing strain was Sphingomonas paucimobilis strain 1443 and the dominant tfdA hybridizing strain was Burkholderia (formerly Pseudomonas) pickettii strain 712, similar to the results of a previous laboratory microcosm study. At the highest 2,4‐D application rate (100 mg kg−1), Southern blots probed by 16S rRNA genes showed a limited number of bands, suggesting that the total soil microbial community had been shifted to one or two dominant strains, presumably 2,4‐D degraders.

Soil bacterial DNA and biovolume profiles measured by flow-cytometry

Flow-cytometry was used to measure cell volumes and DNA contents of single cells in cultures of soil bacteria during exponential growth and starvation conditions. DNA was measured after staining with mitramycin/ethidium bromide. The measurement of DNA was calibrated with rifampicin-treated cells of E. coli containing even numbers of genomes per cell. Cell volumes were assessed by scatter light measurements. Constant DNA to biovolume relations over a range of cell sizes were found for each of the bacteria at exponential growth, and DNA contents per cell varied over a range equivalent to 1–4 genomes per cell. At generation times of 1.0–1.5 h, two genomes were registered as a mean. After starvation of washed cells in a salt solution (24 hrs), a fraction of the cells in each culture had DNA contents equivalent to 1 genome, but significant fractions retained DNA contents equivalent to 2–4 genomes. Attempts to create cells with even numbers of genomes per cell by treatment with rifampicin was successful on an Acinetobacter sp. In contrast, the response to rifampicin was less clear for Pseudomonas fluorescens and P. chlororaphis, and unclear for the gram positive bacteria isolated from soil. The mean decrease in biovolume upon starvation was 4.1 times (range 1.3–8.1 times) and larger than the mean decrease in DNA content of 1.8 (range 1.3–2.7 times). Cell volume determinations by measurements of scatter light was compared with volume determinations by fluorescence microscopy. The amounts of scatter light per volumes was variable, not only did we find large differences between bacterial types, but also between starving and exponentially growing cells of the same isolate. In order to use light scatter as a measure of biovolume, internal standards has to be chosen of comparable size and surface properties as to soil bacteria.

Soil bacterial DNA and biovolume profiles measured by flow-cytometry

Flow-cytometry was used to measure cell volumes and DNA contents of single cells in cultures of soil bacteria during exponential growth and starvation conditions. DNA was measured after staining with mitramycin/ethidium bromide. The measurement of DNA was calibrated with rifampicin-treated cells of E. coli containing even numbers of genomes per cell. Cell volumes were assessed by scatter light measurements. Constant DNA to biovolume relations over a range of cell sizes were found for each of the bacteria at exponential growth, and DNA contents per cell varied over a range equivalent to 1–4 genomes per cell. At generation times of 1.0–1.5 h, two genomes were registered as a mean. After starvation of washed cells in a salt solution (24 hrs), a fraction of the cells in each culture had DNA contents equivalent to 1 genome, but significant fractions retained DNA contents equivalent to 2–4 genomes. Attempts to create cells with even numbers of genomes per cell by treatment with rifampicin was successful on an Acinetobacter sp. In contrast, the response to rifampicin was less clear for Pseudomonas fluorescens and P. chlororaphis, and unclear for the gram positive bacteria isolated from soil. The mean decrease in biovolume upon starvation was 4.1 times (range 1.3–8.1 times) and larger than the mean decrease in DNA content of 1.8 (range 1.3–2.7 times). Cell volume determinations by measurements of scatter light was compared with volume determinations by fluorescence microscopy. The amounts of scatter light per volumes was variable, not only did we find large differences between bacterial types, but also between starving and exponentially growing cells of the same isolate. In order to use light scatter as a measure of biovolume, internal standards has to be chosen of comparable size and surface properties as to soil bacteria.