Clay minerals and metal oxides strongly influence the structure of alkane-degrading microbial communities during soil maturation

Clay minerals, charcoal and metal oxides are essential parts of the soil matrix and strongly influence the formation of biogeochemical interfaces in soil. We investigated the role of these parental materials for the development of functional microbial guilds using the example of alkane-degrading bacteria harbouring the alkane monooxygenase gene (alkB) in artificial mixtures composed of different minerals and charcoal, sterile manure and a microbial inoculum extracted from an agricultural soil. We followed changes in abundance and community structure of alkane-degrading microbial communities after 3 and 12 months of soil maturation and in response to a subsequent 2-week plant litter addition. During maturation we observed an overall increasing divergence in community composition. The impact of metal oxides on alkane-degrading community structure increased during soil maturation, whereas the charcoal impact decreased from 3 to 12 months. Among the clay minerals illite influenced the community structure of alkB-harbouring bacteria significantly, but not montmorillonite. The litter application induced strong community shifts in soils, maturated for 12 months, towards functional guilds typical for younger maturation stages pointing to a resilience of the alkane-degradation function potentially fostered by an extant ‘seed bank''.The tremendous microbial diversity in soils is, among other causes, a result of the huge structural heterogeneity and the enormous variety of biogeochemical interfaces (BGIs) present (Totsche et al., 2010). It has been postulated that during soil development different parental materials shape individual BGIs. They determine to a large extent the specific surface area and charge of BGIs and control for example, the water availability for microorganisms (Young and Crawford, 2004; Chorover et al., 2007). Such abiotic properties of BGIs may strongly influence microbial community structure and function. For instance, Vogel et al. (2014) demonstrated that organic carbon preferentially attaches to rough surfaces of mineral clusters, turning them into hotspots for microbial activity. In this study, we investigated the abundance and community structure of a functional microbial guild (alkane degraders harbouring the alkane monooxygenase gene alkB) at two different time points during the maturation of artificial soils formed from different parental materials. Thereby we compared artificial soils after a shorter period of maturation (namely, 3 months) and more developed soil structures (namely, 12 months of maturation), in each case before and after the addition of alkane-containing substrate (plant litter). We hypothesized that the functional guild studied will be largely influenced by the availability of alkane substrates, essential nutrients (for example, nitrogen) and water, which are largely controlled by differing sorption coefficients of the used parental materials that undergo an alteration process over time. Furthermore we wanted to understand if the ‘dormant or rare biosphere'' could act as a seed bank and ensure process stabilization after substrate addition (Giebler et al., 2013).We used eight artificial soils (Pronk et al., 2012) of identical texture, yet differing in composition regarding the presence of clay minerals (montmorillonite (MT) and illite (IL), metal oxides (ferrihydrite (FH) and boehmite (B)) and charcoal (CH). These materials were chosen as representatives of common soil components with large and reactive surface areas, and can be expected to take part in the formation of BGIs in natural soils (Totsche et al., 2010). Initially, the development of microbial communities had been stimulated by adding a water-extracted fraction of microorganisms from an agricultural soil, classified as a Eutric Cambisol (Ultuna, Sweden) together with sterile manure to the sterile soil components (for details see Supplementary Material S1.1). We took soil samples after maturation phases of either 3 (T3) or 12 months (T12), in which we analysed the abundance (quantitative PCR, qPCR, Supplementary Material S1.2) and community structure (terminal restriction fragment length polymorphism analysis, T-RFLP, Supplementary Material S1.3) of alkane-degrading microbes, as well as response patterns towards the addition of plant litter from winter wheat after 2 weeks of incubation subsequent to maturation. Schulz et al. (2012) described the alkane monooxygenase gene (alkB) as a sensitive marker for the molecular analysis of alkane-degrading bacteria responding to subtle changes in substrate availability that is, fresh plant material, which in general contains large amounts of easily available alkanes in form of waxes. The litter was added on top of the microcosms to create a vertical substrate gradient. Thus, soil samples were taken from the litter–soil interface (top 1 mm of the soil column; T3-I, T12-I) and ‘bulk'' soil (10–11 mm of the soil column; T3-B, T12-B). All details on the Materials and Methods can be found in the Supplementary Material S1.We detected higher alkB gene copy numbers in soils matured for 3 than in those matured for 12 months (Figure 1), regardless whether we analysed them before or after litter addition. Solely the soil where MT and CH had been added together exhibited stable alkB abundance patterns at both time points of maturation. However, the relative response towards litter addition was often stronger in soils with a longer maturation history (T12), particularly when soils contained illite mixtures or ferrihydrite (Figure 1 and Supplementary Material S2). The effects were more pronounced in samples from the litter–soil interface (T3-I and T12-I) than in bulk soil samples (T3-B and T12-B). A previous study revealed that the overall CO₂ respiration rates for these artificial soils after 3 and 12 months of maturation were comparable (Pronk et al., 2012). Several studies therefore concluded that the amount of organic substrates and nutrients present was enough to support activity of the total microflora (Pronk et al., 2012; Babin et al., 2013; Pronk et al., 2013). However, the lower abundance of alkane degraders in soils, maturated for 12 months, in conjunction with the respective stronger response towards litter addition might reflect a depletion of easily bioavailable specific substrates (namely, alkanes) in these soils by degradation and/or maturation-driven reallocation of nutrients to smaller, poorly accessible soil structures (Kögel-Knabner et al., 2008; Pronk et al., 2012; Vogel et al., 2014).Open in a separate windowFigure 1Abundance patterns of alkB genes in artificial soils after 3 (T3) and 12 months (T12). Diamonds (left, y axis) represent alkB gene abundances in matured soils (T3 in black and T12 in grey; s.e. of n=3). Bars (right, y axis) show a relative comparison between the response patterns to litter addition after 12 months compared with 3 months in the different artificial soils (litter–soil interface: black pattern; bulk soil: grey pattern). A value of 1 indicates an identically strong response at T12 and T3 to litter addition, a value >1 indicates a stronger and a value <1 a weaker litter response at T12- compared with T3-litter treated samples. The response values were calculated as quotients of alkB gene abundances after litter treatment vs after maturation, but without litter (for detailed information on calculation see Supplementary Material S1.4; for absolute alkB gene copy numbers of all treatments and correlation with environmental factors see Supplementary Material S2).The community structure of alkB-harbouring bacteria diverged significantly between 3 and 12 months of soil maturation (before litter addition; Figure 2a) with strong differences in some of the dominant T-RFs (for example typically present at T3: 88, 95, 123, 266, 347, 360, 468, 469, 527; more specific for T12: 80, 82, 83, 106; for details see Supplementary Material S1.3 and S3). Moreover, the replicates of the different artificial soil mixtures showed higher variability at T12 when compared with T3 (that is, greater distance between triplicates in the non-metric multidimensional scaling (NMDS)-plot, Figure 2a; P=0.002, statistical calculation Supplementary Material S1.4). This may indicate a higher variability of the alkB community structure with longer maturation time, even within the same artificial soil mixture. When correlating the alkB community structure with the ‘soil complexity'' (that is, the artificial soils contained 1 versus 2 or 3 components in addition to texture-providing quartz), we found that indeed the ‘soil complexity'' significantly influenced the community structure after 3 and 12 months of maturation (P<0.05; Supplementary Material S4-D). Altogether these findings indicate an increasing heterogeneity of the BGIs with maturation time and/or soil complexity, which was accompanied by an increasing formation of microaggregates (Pronk et al., 2012). One may speculate, that the resulting higher hierarchical structure in more matured soils probably creates diverse niches favouring the development of diverging microbial communities in response to their particular microenvironment as also described by Vos et al. (2013).Open in a separate windowFigure 2Community structure of alkB genes in artificial soils illustrated by NMDS. Solid ellipses indicate 95%, dashed ellipses 99% of community spreads of T3 (red) or T12 (blue; for details see Supplementary Material S4). (a) community differences between matured soil without litter at T3 (open symbols) and T12 (closed symbols). The eight artificial soils are indicated by colour (for legend see a; n=1: MT and IL, n=2: FH, n=3: all others). Exemplary for selected soils, distances of replicates to their group centroid are represented by dashed lines, whereby greater distance indicates greater dissimilarity of community structure. (b) alkB community structure before (red or blue ellipse) and after litter addition (green ellipses). The blue arrow indicates strong community shift of T12. (c, d) show selected soil components significantly affecting the alkB communities in all T3 or T12 treatments, respectively (P<at least 0.05; for details see Supplementary Material S4).The addition of fresh plant litter induced a pronounced shift of alkB community patterns in soils, maturated for 12 months (T12-I and T12-B compared with T12; Figure 2b, Supplementary Material S4), whereas no significant changes were observed after 3 months of maturation. Interestingly, the T12-I and T12-B communities after litter addition converged with those of the younger maturation stage, supporting the conclusion that the alkanes might have been depleted during the longer maturation without refeeding. The convergence points to the resilience of the alkane-degradation function through a potential ‘seed bank'' formed by dormant or rare alkane degraders in times of low substrate availability (Giebler et al., 2013). These microbes might be reactivated when the system experiences more favourable environmental conditions (Epstein, 2009; Shade et al., 2012). Their existence is furthermore indicated by high-abundant T-RFs formerly not detected in matured T12 soils (for example, T-RF 86, 88, 94, 95, 239, 463, 523 when comparing T12 with T12-I and T12-B; Supplementary Material S1.3 and S3).When analysing the data sets of 3 and 12 months of maturation individually, the presence of illite and charcoal as soil components in the different soil mixtures were found to be the main factors controlling alkB community structure in all treatments (stronger significance for T3; Figure 2c and Supplementary Material S4) as also observed for the bacterial community in general (Ding et al., 2013). The role of clay minerals like illite might be to sequester large amounts of the soil organic matter (Kaiser and Guggenberger, 2003; Vogel et al., 2014) thereby affecting the nutrient availability (Pronk et al., 2013). The assumption of the reduced availability of nutrients after 3 months is furthermore supported by the significant impact of charcoal, which may adsorb nutrients and reduce their bioavailability (Sohi et al., 2010; Lehmann et al., 2011). A leaching of alkanes by the charcoal used in this study can be excluded (Pronk et al., 2012). However, the charcoal effect receded at later maturation stages, when the reactive sites of minerals and charcoal surfaces gradually became covered with organic matter (Pronk et al., 2012).After 12 months of soil maturation, an emerging relevance of metal oxides as drivers for the community structure of alkB-harbouring microbes (Figure 2e) became evident. Metal oxides strongly interact with the organic matter of natural soils (Eusterhues et al., 2003; Kaiser and Guggenberger, 2003; Wagai and Mayer, 2007). However, a weak affinity and low sorption of organic matter had been reported for our artificial soils owing to constantly neutral pH (Heister et al., 2012; Pronk et al., 2013). Thus, factors other than nutrient availability might be responsible for the influence of metal oxides on the community structure as the increasing heterogeneity of the habitat creates more microniches. Potentially the noncharged surfaces of metal oxides in our experiment might function as valuable ‘safe havens'' still available for colonisation by microbes when other surfaces are already occupied. Thus, their role might become manifest when a soil reaches a higher hierarchical structure.Overall, our results support the hypothesis that complex structured BGIs as generated by clay minerals or metal oxides and charcoal are important drivers for the development of microbial community structures and their functional traits. It seems that structure–diversity and structure–function relationships are emerging properties, which go beyond the characteristics of individual components, that is, they are the result of a dynamic interplay of the BGIs and microbial communities responding to these microhabitats.     

Growth of <Emphasis Type="Italic">Streptomyces</Emphasis> spp. from hydrocarbon-polluted soil on diesel and their analysis for the presence of alkane hydroxylase gene (<Emphasis Type="Italic">alkB</Emphasis>) by PCR

The investigation aimed to examine the Streptomyces flora of hydrocarbon-contaminated soil and study their capability to grow on diesel fuel as a sole carbon source and their analysis for the presence of the alkane hydroxylase gene (alkB) by PCR. A total of 16 Streptomyces isolates were recovered from hydrocarbon-contaminated soil samples on starch casein nitrate agar medium with the ability of 3 isolates to grow on diesel as evaluated by agar plate diffusion method, enzymatic assay and dry weight measurements. PCR analysis of the isolates for the presence of the alkB gene showed two groups with different band size products; group 1 (G1) (316–334 bp) and group 2 (G2) (460–550 bp). Three isolates (SF.1Ac, SF.2Ba, and SF.3Ad) grew around diesel-containing wells and contained the alkB gene with size band ranged between 320 and 550 bp. However; one isolate (SF.1Aa) did not show any PCR product although it was able to grow on diesel. This implies that the alkB gene is not the only gene that is responsible for the degradation of alkanes. Further, the variation in the G2 fragment size probably indicates different related genes that might be involved in alkane degradation rather than a single gene.     

Changes in Microbial Community Composition and Function during a Polyaromatic Hydrocarbon Phytoremediation Field Trial

The purpose of this study was to investigate the mechanism by which phytoremediation systems promote hydrocarbon degradation in soil. The composition and degradation capacity of the bulk soil microbial community during the phytoremediation of soil contaminated with aged hydrocarbons was assessed. In the bulk soil, the level of catabolic genes involved in hydrocarbon degradation (ndoB, alkB, and xylE) as well as the mineralization of hexadecane and phenanthrene was higher in planted treatment cells than in treatment cells with no plants. There was no detectable shift in the 16S ribosomal DNA (rDNA) composition of the bulk soil community between treatments, but there were plant-specific and -selective effects on specific catabolic gene prevalence. Tall Fescue (Festuca arundinacea) increased the prevalence of ndoB, alkB, and xylE as well as naphthalene mineralization in rhizosphere soil compared to that in bulk soil. In contrast, Rose Clover (Trifolium hirtum) decreased catabolic gene prevalence and naphthalene mineralization in rhizosphere soil. The results demonstrated that phytoremediation systems increase the catabolic potential of rhizosphere soil by altering the functional composition of the microbial community. This change in composition was not detectable by 16S rDNA but was linked to specific functional genotypes with relevance to petroleum hydrocarbon degradation.     

Selection of Specific Endophytic Bacterial Genotypes by Plants in Response to Soil Contamination

Plant-bacterial combinations can increase contaminant degradation in the rhizosphere, but the role played by indigenous root-associated bacteria during plant growth in contaminated soils is unclear. The purpose of this study was to determine if plants had the ability to selectively enhance the prevalence of endophytes containing pollutant catabolic genes in unrelated environments contaminated with different pollutants. At petroleum hydrocarbon contaminated sites, two genes encoding hydrocarbon degradation, alkane monooxygenase (alkB) and naphthalene dioxygenase (ndoB), were two and four times more prevalent in bacteria extracted from the root interior (endophytic) than from the bulk soil and sediment, respectively. In field sites contaminated with nitroaromatics, two genes encoding nitrotoluene degradation, 2-nitrotoluene reductase (ntdAa) and nitrotoluene monooxygenase (ntnM), were 7 to 14 times more prevalent in endophytic bacteria. The addition of petroleum to sediment doubled the prevalence of ndoB-positive endophytes in Scirpus pungens, indicating that the numbers of endophytes containing catabolic genotypes were dependent on the presence and concentration of contaminants. Similarly, the numbers of alkB- or ndoB-positive endophytes in Festuca arundinacea were correlated with the concentration of creosote in the soil but not with the numbers of alkB- or ndoB-positive bacteria in the bulk soil. Our results indicate that the enrichment of catabolic genotypes in the root interior is both plant and contaminant dependent.     

Weeds influence soil bacterial and fungal communities

Background and aims

Vineyards harbour a variety of weeds, which are usually controlled since they compete with grapevines for water and nutrients. However, weed plants may host groups of fungi and bacteria exerting important functions.

Methods

We grew three different common vineyard weeds (Taraxacum officinalis, Trifolium repens and Poa trivialis) in four different soils to investigate the effects of weeds and soil type on bacterial and fungal communities colonising bulk soil, rhizosphere and root compartments. Measurements were made using the cultivation-independent technique Automated Ribosomal Intergenic Spacer Analysis (ARISA).

Results

Weeds have a substantial effect on roots but less impact on the rhizosphere and bulk soil, while soil type affects all three compartments, in particular the bulk soil community. The fungal, but not the bacterial, bulk soil community structure was affected by the plants at the late experimental stage. Root communities contained a smaller number of Operational Taxonomic Units (OTUs) and different bacterial and fungal structures compared with rhizosphere and bulk soil communities.

Conclusions

Weed effect is localised to the rhizosphere and does not extend to bulk soil in the case of bacteria, although the structure of fungal communities in the bulk soil may be influenced by some weed plants.     

The Use of a Combination of alkB Primers to Better Characterize the Distribution of Alkane-Degrading Bacteria

The alkane monooxygenase AlkB, which is encoded by the alkB gene, is a key enzyme involved in bacterial alkane degradation. To study the alkB gene within bacterial communities, researchers need to be aware of the variations in alkB nucleotide sequences; a failure to consider the sequence variations results in the low representation of the diversity and richness of alkane-degrading bacteria. To minimize this shortcoming, the use of a combination of three alkB-targeting primers to enhance the detection of the alkB gene in previously isolated alkane-degrading bacteria was proposed. Using this approach, alkB-related PCR products were detected in 79% of the strains tested. Furthermore, the chosen set of primers was used to study alkB richness and diversity in different soils sampled in Carmópolis, Brazil and King George Island, Antarctica. The DNA extracted from the different soils was PCR amplified with each set of alkB-targeting primers, and clone libraries were constructed, sequenced and analyzed. A total of 255 alkB phylotypes were detected. Venn diagram analyses revealed that only low numbers of alkB phylotypes were shared among the different libraries derived from each primer pair. Therefore, the combination of three alkB-targeting primers enhanced the richness of alkB phylotypes detected in the different soils by 45% to 139%, when compared to the use of a single alkB-targeting primer. In addition, a dendrogram analysis and beta diversity comparison of the alkB composition showed that each of the sampling sites studied had a particular set of alkane-degrading bacteria. The use of a combination of alkB primers was an efficient strategy for enhancing the detection of the alkB gene in cultivable bacteria and for better characterizing the distribution of alkane-degrading bacteria in different soil environments.     

Alkane‐degrading properties of Dietzia sp. H0B,a key player in the Prestige oil spill biodegradation (NW Spain)

Aims: Investigation of the alkane‐degrading properties of Dietzia sp. H0B, one of the isolated Corynebacterineae strains that became dominant after the Prestige oil spill. Methods and Results: Using molecular and chemical analyses, the alkane‐degrading properties of strain Dietzia sp. H0B were analysed. This Grampositive isolate was able to grow on n‐alkanes ranging from C₁₂ to C₃₈ and branched alkanes (pristane and phytane). 8‐Hexadecene was detected as an intermediate of hexadecane degradation by Dietzia H0B, suggesting a novel alkane‐degrading pathway in this strain. Three putative alkane hydroxylase genes (one alkB homologue and two CYP153 gene homologues of cytochrome P450 family) were PCR‐amplified from Dietzia H0B and differed from previously known hydroxylase genes, which might be related to the novel degrading activity observed on Dietzia H0B. The alkane degradation activity and the alkB and CYP153 gene expression were observed constitutively regardless of the presence of the substrate, suggesting additional, novel pathways for alkane degradation. Conclusions: The results from this study suggest novel alkane‐degrading pathways in Dietzia H0B and a genetic background coding for two different putative oil‐degrading enzymes, which is mostly unexplored and worth to be subject of further functional analysis. Significance and Impact of the Study: This study increases the scarce information available about the genetic background of alkane degradation in genus Dietzia and suggests new pathways and novel expression mechanisms of alkane degradation.     

Abundance and Diversity of <Emphasis Type="Italic">n</Emphasis>-Alkane-Degrading Bacteria in a Forest Soil Co-Contaminated with Hydrocarbons and Metals: A Molecular Study on <Emphasis Type="Italic">alkB</Emphasis> Homologous Genes

Unraveling functional genes related to biodegradation of organic compounds has profoundly improved our understanding of biological remediation processes, yet the ecology of such genes is only poorly understood. We used a culture-independent approach to assess the abundance and diversity of bacteria catalyzing the degradation of n-alkanes with a chain length between C₅ and C₁₆ at a forest site co-contaminated with mineral oil hydrocarbons and metals for nearly 60 years. The alkB gene coding for a rubredoxin-dependent alkane monooxygenase enzyme involved in the initial activation step of aerobic aliphatic hydrocarbon metabolism was used as biomarker. Within the area of study, four different zones were evaluated: one highly contaminated, two intermediately contaminated, and a noncontaminated zone. Contaminant concentrations, hydrocarbon profiles, and soil microbial respiration and biomass were studied. Abundance of n-alkane-degrading bacteria was quantified via real-time PCR of alkB, whereas genetic diversity was examined using molecular fingerprints (T-RFLP) and clone libraries. Along the contamination plume, hydrocarbon profiles and increased respiration rates suggested on-going natural attenuation at the site. Gene copy numbers of alkB were similar in contaminated and control areas. However, T-RFLP-based fingerprints suggested lower diversity and evenness of the n-alkane-degrading bacterial community in the highly contaminated zone compared to the other areas; both diversity and evenness were negatively correlated with metal and hydrocarbon concentrations. Phylogenetic analysis of alkB denoted a shift of the hydrocarbon-degrading bacterial community from Gram-positive bacteria in the control zone (most similar to Mycobacterium and Nocardia types) to Gram-negative genotypes in the contaminated zones (Acinetobacter and alkB sequences with little similarity to those of known bacteria). Our results underscore a qualitative rather than a quantitative response of hydrocarbon-degrading bacteria to the contamination at the molecular level.     

Novel Alkane Hydroxylase Gene (alkB) Diversity in Sediments Associated with Hydrocarbon Seeps in the Timor Sea,Australia

Hydrocarbon seeps provide inputs of petroleum hydrocarbons to widespread areas of the Timor Sea. Alkanes constitute the largest proportion of chemical components found in crude oils, and therefore genes involved in the biodegradation of these compounds may act as bioindicators for this ecosystem''s response to seepage. To assess alkane biodegradation potential, the diversity and distribution of alkane hydroxylase (alkB) genes in sediments of the Timor Sea were studied. Deduced AlkB protein sequences derived from clone libraries identified sequences only distantly related to previously identified AlkB sequences, suggesting that the Timor Sea maybe a rich reservoir for novel alkane hydroxylase enzymes. Most sequences clustered with AlkB sequences previously identified from marine Gammaproteobacteria though protein sequence identities averaged only 73% (with a range of 60% to 94% sequence identities). AlkB sequence diversity was lower in deep water (>400 m) samples off the continental slope than in shallow water (<100 m) samples on the continental shelf but not significantly different in response to levels of alkanes. Real-time PCR assays targeting Timor Sea alkB genes were designed and used to quantify alkB gene targets. No correlation was found between gene copy numbers and levels of hydrocarbons measured in sediments using sensitive gas chromatography-mass spectrometry techniques, probably due to the very low levels of hydrocarbons found in most sediment samples. Interestingly, however, copy numbers of alkB genes increased substantially in sediments exposed directly to active seepage even though only low or undetectable concentrations of hydrocarbons were measured in these sediments in complementary geochemical analyses due to efficient biodegradation.Alkanes are saturated hydrocarbons that are widespread in marine environments due to a variety of anthropogenic and natural sources. They constitute the major fraction of hydrocarbon components found in crude oils and refined petroleum and are also produced by various marine organisms (e.g., zooplankton) as cellular components (2, 44). Alkanes are considered as pollutants, with short-chained alkanes acting as solvents toward cellular membranes and other lipid components (34) while longer-chained alkanes may contribute to the formation of oil films and slicks that may limit nutrient and oxygen exchange (21). Importantly, alkanes also serve as important carbon and energy sources for some microorganisms. In marine environments, alkanes succumb to various removal and dispersal processes such as dissolution, photochemical oxidation, evaporation, adsorption, and sedimentation. However, the greatest removal pathway for alkanes in marine sediments is via biodegradation by bacteria (13). This mechanism also mediates the transfer of oil-derived carbon to higher trophic levels (28, 37), and therefore these bacteria have an important role in carbon cycling in environments subject to long-term inputs of hydrocarbons such as marine seep-associated ecosystems. Alkane biodegradation is mediated by a diverse range of marine bacteria using various electron acceptors although degradation generally proceeds at greater rates under aerobic conditions than under anaerobic conditions, where the process is relatively slow (8, 26).In the presence of oxygen, well-characterized alkane oxidation pathways are initiated by an activation step whereby oxygen is introduced to the alkane substrate before further catabolic steps can proceed. A number of oxygen-dependent alkane hydroxylase enzyme systems have been discovered that catalyze this initial step including the soluble di-iron methane monooxygenases and the membrane-bound copper-containing methane monooxygenases, both of which act upon short-chain alkanes (i.e., C₁ up to C₈). Integral membrane non-heme iron alkane hydroxylases (the alk system) that are related to the well-characterized AlkB of Pseudomonas putida GPo1 (also known as Pseudomonas oleovorans TF4-1 I) act upon longer-chain alkanes (i.e., C₅ to C₁₆) (40). Other systems exist that include alkane-hydroxylating cytochrome P450 enzymes in addition to other enzyme systems that are known to exist based purely on chemical analyses of metabolites formed during alkane degradation experiments (22, 25, 29); however, knowledge pertaining to the enzymes and genes involved as well as their importance in the environment is limited. Only recently have genes involved in the degradation of long-chain alkanes (e.g., C₃₂ and C₃₆) been identified in Acinetobacter sp. strain DSM 17874 (39) though there is no information about the presence or importance of such enzymes in the environment.Although various chemical and microbiological aspects of petroleum oil and alkane biodegradation in marine systems have been relatively well studied, there is a general lack of knowledge concerning the diversity or abundance of the functional genes involved. The biochemical and molecular aspects of alkB genes and the enzymes they encode have been relatively well studied, and this has enabled the development of molecular tools for the study of alkB genes in the environment (19). Elevated levels of hydrocarbons or the introduction of hydrocarbons to environments has been shown to increase gene copy numbers, indicating the potential use of alkB genes as bioindicators of oil pollution and/or biodegradation (16, 33, 36, 43). However, to date only one study has used culture-independent molecular methods to examine the diversity of alkB genes in a marine environment (20), and no studies have examined hydrocarbon-degrading genes where natural hydrocarbon seepage occurs.In this study, the diversity and relative abundance of alkB genes were examined in sediments of the Timor Sea, a region where natural seeps are sources of widespread petroleum hydrocarbons. It was hypothesized that (i) novel alkB genes may exist in this unique tropical marine environment, (ii) that variations in gene diversity would be found in sediments with different hydrocarbon levels, and (iii) that the abundance of certain alkB gene types may reflect the levels of measured hydrocarbons in sediments, and therefore this assay could be used as a complementary tool for monitoring petroleum inputs into sediments of the Timor Sea.     

Recovery of microbial diversity and activity during bioremediation following chemical oxidation of diesel contaminated soils

To improve the coupling of in situ chemical oxidation and in situ bioremediation, a systematic analysis was performed of the effect of chemical oxidation with Fenton's reagent, modified Fenton's reagent, permanganate, or persulfate, on microbial diversity and activity during 8 weeks of incubation in two diesel-contaminated soils (peat and fill). Chemical oxidant and soil type affected the microbial community diversity and biodegradation activity; however, this was only observed following treatment with Fenton's reagent and modified Fenton's reagent, and in the biotic control without oxidation. Differences in the highest overall removal efficiencies of 69 % for peat (biotic control) and 59 % for fill (Fenton's reagent) were partially explained by changes in contaminant soil properties upon oxidation. Molecular analysis of 16S rRNA and alkane monooxygenase (alkB) gene abundances indicated that oxidation with Fenton's reagent and modified Fenton's reagent negatively affected microbial abundance. However, regeneration occurred, and final relative alkB abundances were 1–2 orders of magnitude higher in chemically treated microcosms than in the biotic control. 16S rRNA gene fragment fingerprinting with DGGE and prominent band sequencing illuminated microbial community composition and diversity differences between treatments and identified a variety of phylotypes within Alpha-, Beta-, and Gammaproteobacteria. Understanding microbial community dynamics during coupled chemical oxidation and bioremediation is integral to improved biphasic field application.     

The influence of litter quality and micro-habitat on litter decomposition and soil properties in a silvopasture system

Studies to understand litter processes and soil properties are useful for maintaining pastureland productivity as animal husbandry is the dominant occupation in the hot arid region. We aimed to quantify how micro-habitats and combinations of litters of the introduced leguminous tree Colophospermum mopane with the grasses Cenchrus ciliaris or Lasiurus sindicus influence decomposition rate and soil nutrient changes in a hot desert silvopasture system. Litter bags with tree litter alone (T), tree + C. ciliaris in 1:1 ratio (TCC) and tree + L. sindicus 1:1 ratio (TLS) litter were placed inside and outside of the C. mopane canopy and at the surface, 3–7 cm and 8–12 cm soil depths. We examined litter loss, soil fauna abundance, organic carbon (SOC), total (TN), ammonium (NH₄–N) and nitrate (NO₃–N) nitrogen, phosphorus (PO₄–P), soil respiration (SR) and dehydrogenase activity (DHA) in soil adjacent to each litter bag. After 12 months exposure, the mean residual litter was 40.2% of the initial value and annual decomposition rate constant (k) was 0.98 (0.49–1.80). Highest (p < 0.01) litter loss was in the first four months, when faunal abundance, SR, DHA and humidity were highest but it decreased with time. These variables and k were highest under the tree canopies. The litter loss and k were highest (p < 0.01) in TLS under the tree canopy, but the reverse trend was found for litter outside the canopy. Faunal abundance, litter loss, k, nutrient release and biochemical activities were highest (p < 0.01) in the 3–7 cm soil layer. Positive correlations of litter loss and soil fauna abundance with soil nutrients, SR and DHA demonstrated the interactions of litter quality and micro-habitats together with soil fauna on increased soil fertility. These results suggest that a Colophospermum mopane and L. sindicus silvopasture system best promotes faunal abundance, litter decomposition and soil fertility. The properties of these species and the associated faunal resources may be utilised as an ecosystem-restoration strategy in designing a silvopasture system. This may help to control land degradation and increase productivity sustainably in this environment.     

北京东灵山土壤动物多样性海拔格局的尺度推绎规律

识别群落内部各类群多样性格局的复杂性是生态学家面临的挑战,而尺度推绎规律是揭示复杂生态结构的有效途径之一。本研究利用多重分形的方法探索了不同海拔土壤动物多样性格局的尺度推绎规律,对比分析了凋落物层和土壤层之间多重分形谱的差异。结果表明: 与之前对植物群落的分析结果相似,土壤动物多样性尺度推绎规律同样具有幂律特征,如丰富度、Shannon多样性指数和Simpson多样性的倒数。凋落物层和土壤层中不同相对多度土壤动物的丰富度也具有幂律尺度推绎规律。凋落物层和土壤层中土壤动物多样性格局都具有多重分形特征,但凋落物层中多样性的分形结构比土壤层更均匀,且两层间优势类群与稀有类群的尺度推绎特征在多重分形谱上不同格局。幂律尺度推绎规律对于有着较高丰富度与多度的土壤动物同样存在,从而有助于揭示地下生物多样性的空间分布机制。     

三种温带森林大型土壤动物群落结构的时空动态

对帽儿山3种典型森林群落大型土壤动物进行了连续6个月的野外调查研究。通过系统分析,共获得大型土壤动物3604只,隶属于3门6纲17目50科。其中正蚓科(Lumbricidae)、线蚓科(Enchytraeidae)和石蜈蚣目(Lithobiomorpha)为优势类群,常见类群11类。结果表明:(1)水平分布上,密度和生物量红松人工林最高,其次为硬阔叶林,蒙古栎林最少;类群数硬阔叶林最多,蒙古栎林最少;香农指数和丰富度指数均为蒙古栎林最高,红松人工林最低;优势度指数与两者相反;均匀度指数蒙古栎林最高,硬阔叶林最低;(2)垂直分布上,个体密度、类群数及生物量均差异显著(P < 0.001)。3个样地大型土壤动物个体密度表聚性明显;类群数红松人工林自凋落物层向下减少,硬阔叶林和蒙古栎林0-10 cm最多;生物量在0-10 cm土层最大;香农指数随深度增加而减小,优势度指数则相反;(3)在时间变化上,5月和10月个体密度和类群数较多,9月生物量最大;香农指数和优势度指数差异显著(P < 0.01),其他指数各月间无明显差异;(4)与土壤环境因子关系上,总有机碳含量与类群数、个体密度及生物量显著正相关,容重与香农指数显著负相关;典型对应分析结果表明,不同类群大型土壤动物与环境相关性不同。     

Microaerophilic alkane degradation in <Emphasis Type="Italic">Pseudomonas extremaustralis</Emphasis>: a transcriptomic and physiological approach

Diesel fuel is one of the most important sources of hydrocarbon contamination worldwide. Its composition consists of a complex mixture of n-alkanes, branched alkanes and aromatic compounds. Hydrocarbon degradation in Pseudomonas species has been mostly studied under aerobic conditions; however, a dynamic spectrum of oxygen availability can be found in the environment. Pseudomonas extremaustralis, an Antarctic bacterium isolated from a pristine environment, is able to degrade diesel fuel and presents a wide microaerophilic metabolism. In this work RNA-deep sequence experiments were analyzed comparing the expression profile in aerobic and microaerophilic cultures. Interestingly, genes involved in alkane degradation, including alkB, were over-expressed in micro-aerobiosis in absence of hydrocarbon compounds. In minimal media supplemented with diesel fuel, n-alkanes degradation (C13–C19) after 7 days was observed under low oxygen conditions but not in aerobiosis. In-silico analysis of the alkB promoter zone showed a putative binding sequence for the anaerobic global regulator, Anr. Our results indicate that some diesel fuel components can be utilized as sole carbon source under microaerophilic conditions for cell maintenance or slow growth in a Pseudomonas species and this metabolism could represent an adaptive advantage in polluted environments.     

Analysis of Genes for Succinoyl Trehalose Lipid Production and Increasing Production in Rhodococcus sp. Strain SD-74

Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus species bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl coenzyme A (acyl-CoA) transferase (tlsA), fructose-bisphosphate aldolase (fda), and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis, and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. strain SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in Rhodococcus species.     

Identification of nitrogen-incorporating bacteria in petroleum-contaminated arctic soils by using [15N]DNA-based stable isotope probing and pyrosequencing

Arctic soils are increasingly susceptible to petroleum hydrocarbon contamination, as exploration and exploitation of the Arctic increase. Bioremediation in these soils is challenging due to logistical constraints and because soil temperatures only rise above 0°C for ∼2 months each year. Nitrogen is often added to contaminated soil in situ to stimulate the existing microbial community, but little is known about how the added nutrients are used by these microorganisms. Microbes vary widely in their ability to metabolize petroleum hydrocarbons, so the question becomes: which hydrocarbon-degrading microorganisms most effectively use this added nitrogen for growth? Using [¹⁵N]DNA-based stable isotope probing, we determined which taxonomic groups most readily incorporated nitrogen from the monoammonium phosphate added to contaminated and uncontaminated soil in Canadian Forces Station-Alert, Nunavut, Canada. Fractions from each sample were amplified with bacterial 16S rRNA and alkane monooxygenase B (alkB) gene-specific primers and then sequenced using lage-scale parallel-pyrosequencing. Sequence data was combined with 16S rRNA and alkB gene C quantitative PCR data to measure the presence of various phylogenetic groups in fractions at different buoyant densities. Several families of Proteobacteria and Actinobacteria that are directly involved in petroleum degradation incorporated the added nitrogen in contaminated soils, but it was the DNA of Sphingomonadaceae that was most enriched in ¹⁵N. Bacterial growth in uncontaminated soils was not stimulated by nutrient amendment. Our results suggest that nitrogen uptake efficiency differs between bacterial groups in contaminated soils. A better understanding of how groups of hydrocarbon-degraders contribute to the catabolism of petroleum will facilitate the design of more targeted bioremediation treatments.     

四川盆地亚热带常绿阔叶林不同物候期凋落物分解与土壤动物群落结构的关系

为了解植物生长不同物候时期凋落物分解过程中土壤动物群落结构动态及其与凋落物分解的关系,以四川盆地亚热带常绿阔叶林典型人工林树种马尾松和柳杉,次生林树种香樟和麻栎凋落物为研究对象,采用凋落物分解袋试验研究,凋落物分解过程中土壤动物的群落特征。4种凋落物分解袋共获得土壤动物8047只,其中,柳杉(2341只)>香樟(2105只)>马尾松(2046只)>麻栎(1555只)。其中,秋末落叶期、萌动期和展叶期,马尾松凋落物袋中主要以捕食性土壤动物为优势类群,而后以菌食性土壤动物为主;香樟凋落物袋中除秋末落叶期和叶衰期以菌食性土壤动物为主要优势类群外,其他各时期均以捕食性土壤动物为主要优势类群;柳杉凋落物分解各时期均以菌食性土壤动物为主要优势类群;麻栎凋落物分解在前3个时期以菌食性为主,而后以植食性土壤动物为主要优势类群。相关分析表明,在秋末落叶期和萌动期土壤动物的个体密度主要和氮、磷含量及其格局密切相关,叶衰期主要和难分解组分木质素显著相关。除在秋末落叶期土壤动物对凋落物分解的贡献率与土壤动物的个体密度显著相关外,其余主要物候关键时期均与土壤动物的类群密度及其食性显著相关。     

鄂尔多斯高原弃耕农田恢复过程中土壤物理性质和生物结皮的变化

在鄂尔多斯高原软梁、硬梁弃耕农田上选取一系列不同时限的弃耕农田,研究弃耕演替过程中地表凋落物、生物结皮,以及不同土层土壤容重、机械组成、含水量的动态变化.结果表明: 软梁弃耕农田表层(0～10 cm)物理环境随弃耕年限增加明显改善,土壤黏粒含量、地表凋落物随弃耕年限增加明显增加,土壤容重明显降低,土壤含水率略微升高;中层(10～30 cm)土壤物理环境随弃耕年限增加稍显恶化,土壤黏粒含量升高,土壤含水量略微降低;深层(30～50 cm)土壤物理性质变异较大.硬梁弃耕农田土壤表面凋落物盖度、生物结皮盖度、生物结皮厚度随弃耕年限增加而增加,表层(0～10 cm)土壤容重、黏粒含量、含水量随弃耕年限变化总体变化不显著,深层(10～40 cm)土壤物理性质变异较大.地表凋落物和土壤黏粒物质的累积导致软梁弃耕农田土壤水分浅层化,可能是20年内浅根性多年生草本植物群落取代半灌木油蒿群落的关键因素.硬梁弃耕农田在演替过程中未见油蒿入侵,可能与较高的浅层含水量和生物结皮发育有关.     

Biodegradation of aged diesel in diverse soil matrixes: impact of environmental conditions and bioavailability on microbial remediation capacity

While bioremediation of total petroleum hydrocarbons (TPH) is in general a robust technique, heterogeneity in terms of contaminant and environmental characteristics can impact the extent of biodegradation. The current study investigates the implications of different soil matrix types (anthropogenic fill layer, peat, clay, and sand) and bioavailability on bioremediation of an aged diesel contamination from a heterogeneous site. In addition to an uncontaminated sample for each soil type, samples representing two levels of contamination (high and low) were also used; initial TPH concentrations varied between 1.6 and 26.6 g TPH/kg and bioavailability between 36 and 100 %. While significant biodegradation occurred during 100 days of incubation under biostimulating conditions (64.4–100 % remediation efficiency), low bioavailability restricted full biodegradation, yielding a residual TPH concentration. Respiration levels, as well as the abundance of alkB, encoding mono-oxygenases pivotal for hydrocarbon metabolism, were positively correlated with TPH degradation, demonstrating their usefulness as a proxy for hydrocarbon biodegradation. However, absolute respiration and alkB presence were dependent on soil matrix type, indicating the sensitivity of results to initial environmental conditions. Through investigating biodegradation potential across a heterogeneous site, this research illuminates the interplay between soil matrix type, bioavailability, and bioremediation and the implications of these parameters for the effectiveness of an in situ treatment.     

Cloning and expression of three ladA-type alkane monooxygenase genes from an extremely thermophilic alkane-degrading bacterium Geobacillus thermoleovorans B23

An extremely thermophilic bacterium, Geobacillus thermoleovorans B23, is capable of degrading a broad range of alkanes (with carbon chain lengths ranging between C11 and C32) at 70 °C. Whole-genome sequence analysis revealed that unlike most alkane-degrading bacteria, strain B23 does not possess an alkB-type alkane monooxygenase gene. Instead, it possesses a cluster of three ladA-type genes, ladAα_B23, ladAβ_B23, and ladB _B23, on its chromosome, whose protein products share significant amino acid sequence identities, 49.8, 34.4, and 22.7 %, respectively, with that of ladA alkane monooxygenase gene found on a plasmid of Geobacillus thermodetrificans NG 80-2. Each of the three genes, ladAα_B23, ladAβ_B23, and ladB _B23, was heterologously expressed individually in an alkB1 deletion mutant strain, Pseudomonas fluorescens KOB2Δ1. It was found that all three genes were functional in P. fluorescens KOB2Δ1, and partially restored alkane degradation activity. In this study, we suggest that G. thermoleovorans B23 utilizes multiple LadA-type alkane monooxygenases for the degradation of a broad range of alkanes.