Early detection of viable Francisella tularensis in environmental matrices by culture-based PCR.

BACKGROUND: Francisella tularensis is a fastidious, Gram-negative coccobacillus and is the causative agent of tularemia. To assess viability yet overcome lengthy incubation periods, a culture-based PCR method was used to detect early growth of the lowest possible number of F. tularensis cells. This method utilized a previously developed enhanced F. tularensis growth medium and is based on the change in PCR cycle threshold at the start and end of each incubation. RESULTS: To test method robustness, a virulent Type A1 (Schu4) and B (IN99) strain and the avirulent Live Vaccine Strain (LVS) were incubated with inactivated target cells, humic acid, drinking and well water, and test dust at targeted starting concentrations of 1, 10, and 100 CFU mL- 1 (low, mid, and high, respectively). After 48 h, LVS growth was detected at all targeted concentrations in the presence of 106 inactivated LVS cells; while Schu4 and IN99 growth was detected in the presence of 104 Schu4 or IN99 inactivated cells at the mid and high targets. Early detection of F. tularensis growth was strain and concentration dependent in the presence of fast-growing well water and test dust organisms. In contrast, growth was detected at each targeted concentration by 24 h in humic acid and drinking water for all strains. CONCLUSIONS: Results indicated that the culture-based PCR assay is quick, sensitive, and specific while still utilizing growth as a measure of pathogen viability. This method can circumvent lengthy incubations required for Francisella identification, especially when swift answers are needed during epidemiological investigations, remediation efforts, and decontamination verification.

Removing organic matters from reverse osmosis concentrate using advanced oxidation-biological activated carbon process combined with Fe3+/humus-reducing bacteria.

The high-concentration wastewater produced in the industrial reverse osmosis (RO) process contains a large amount of refractory organic matters, which will have serious impacts on the natural environment and human health. Among them, contaminants can be transformed by humus-reducing bacteria based on humus. In this study, O3- assisted UV-Fenton method was applied as pretreatment. Biological activated carbon (BAC) technology in which humus-reducing bacteria were the dominant bacteria, enhanced by electron donor and Fe3+, was used to dispose of RO concentrate (ROC). The results showed that water treatment process combining oxidation with biological filtration had a positive effect on the removal of stubborn contaminants in ROC. The system was strengthened by adding electron donor and Fe3+, and the chemical oxygen demand (COD) removal efficiency was up to 80.1%. However, when the removal efficiency of UV254 absorbing pollutants reached optimal value (87.3%), that means only Fe3+ was added.

Effects of organic matter and low oxygen on the mycobenthos in a coastal lagoon.

Fungi living in sediments ('mycobenthos') are hypothesized to play a role in the degradation of organic matter deposited at the land-sea interface, but the environmental factors influencing the mycobenthos are poorly understood. We used mock community calibrated Illumina sequencing to show that the mycobenthos community structure in a coastal lagoon was significantly changed after exposure to a lignocellulose extract and subsequent development of benthic anoxia over a relatively short (10 h) incubation. Saprotrophic taxa dominated and were selected for under benthic anoxia, specifically Aquamyces (Chytridiomycota) and Orbilia (Ascomycota), implicating these genera as important benthic saprotrophs. Protein encoding genes involved in energy and biomass production from Fungi and the fungal-analogue group Labyrinthulomycetes had the highest increase in expression with the added organic matter compared with all other groups, indicating that lignocellulose stimulates metabolic activity in the mycobenthos. Flavobacteria dominated the active bacterial community that grew rapidly with the lignocellulose extract and crashed sharply upon O2 depletion. Our findings indicate that the diversity, activity and trophic potential of the mycobenthos changes rapidly in response to organic matter and decreasing O2 concentrations, which together with heterotrophic Flavobacteria, undergo 'boom and bust' dynamics during lignocellulose degradation in estuarine ecosystems.

Xinfangfangia humi sp. nov., isolated from soil amended with humic acid.

Two slightly beige-pigmented, Gram-stain-negative, rod-shaped bacterial strains, IMT-291T and IMT-297, were isolated from soil in a field located in Malvern, Alabama, USA. The source soil had been amended with humic acid and continuously used for the cultivation of worms used for fish bait. It is still conceivable that the source of the strains is from the humic acid amendment, although all attempts to isolate the novel phenotypes from the humic acid source have failed. The two strains were identical based on morphology, growth rate and subsequently by 16S rRNA gene sequences, but showed differences in genomic fingerprint patterns generated by rep-PCR. Phylogenetic analysis based on the 16S rRNA gene revealed a placement of the strain in a distinct cluster with Xinfangfangia soli (97.2 % 16S rRNA gene sequence similarity) and in close proximity to the genus Falsirhodobacter with highest 16S rRNA gene sequence similarity of 95.3 % to the type strain of Falsirhodobacter deserti. Sequence similarities to all other type strains were below 95.0 %. The chemotaxonomic analysis showed a clear similarity to the genus Xinfangfangia. The main cellular fatty acids of the strain were C18 : 1 ω7c, 11-methly-C18 : 1 ω7c and C16 : 0. The major quinone was ubiquinone Q-10. Phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol and phosphatidylcholine were predominant in the polar lipid profile. The polyamine pattern contained the major compound spermidine and moderate amounts of putrescine and cadaverine. The diamino acid of the peptidoglycan was meso-diaminopimelic acid. Based on phylogenetic, chemotaxonomic and phenotypic analyses we propose a new species of the genus Xinfangfangia, with the name Xinfangfangiahumi sp. nov. and strain IMT-291T (=LMG 30636T=CIP 111625T=CCM 8858T) as type strain.

Microbial Organic Matter Utilization in High-Arctic Streams: Key Enzymatic Controls.

In the Arctic, climate changes contribute to enhanced mobilization of organic matter in streams. Microbial extracellular enzymes are important mediators of stream organic matter processing, but limited information is available on enzyme processes in this remote area. Here, we studied the variability of microbial extracellular enzyme activity in high-Arctic fluvial biofilms. We evaluated 12 stream reaches in Northeast Greenland draining areas exhibiting different geomorphological features with contrasting contents of soil organic matter to cover a wide range of environmental conditions. We determined stream nitrogen, phosphorus, and dissolved organic carbon concentrations, quantified algal biomass and bacterial density, and characterized the extracellular enzyme activities involved in catalyzing the cleavage of a range of organic matter compounds (e.g., ß-glucosidase, phosphatase, ß-xylosidase, cellobiohydrolase, and phenol oxidase). We found significant differences in microbial organic matter utilization among the study streams draining contrasting geomorphological features, indicating a strong coupling between terrestrial and stream ecosystems. Phosphatase and phenol oxidase activities were higher in solifluction areas than in alluvial areas. Besides dissolved organic carbon, nitrogen availability was the main driver controlling enzyme activities in the high-Arctic, which suggests enhanced organic matter mineralization at increased nutrient availability. Overall, our study provides novel information on the controls of organic matter usage by high-Arctic stream biofilms, which is of high relevance due to the predicted increase of nutrient availability in high-Arctic streams in global climate change scenarios.

Microbially reduced humic acid promotes the anaerobic photodegradation of 17α--ethinylestradiol.

Photolysis and microbial activity are relatively obvious in shallow, eutrophic waters with low dissolved oxygen content. Ubiquitous humic acid (HA) can act as electron acceptor and be reduced by bacterial under such conditions, and the reduced form of humic acid (RHA) plays an important role in the photolysis contaminants. In this study, anaerobic 17α-ethinylestradiol (EE2) photodegradation was performed along with biodegradation by Shewanella putrefaciens mediated by HA. The mechanism of such coupled photolysis and biodegradation of EE2 was thus elucidated. The removal rate in such coupled degradation in the presence of 10 mgC L-1 of HA at pH 8.0 was greater than that of either photolysis or biodegradation alone. HA which had been reduced in a double-chamber microbial fuel cell showed better promotion to EE2 photodegradation than fresh HA. Reactive species scavenging experiments indicated that hydroxyl radical and excited triplet states of HA were primary contributors to EE2 photodegradation in anaerobic conditions. More of them were produced from RHA than from pristine HA. Besides, the degraded EE2 solutions inhibited the proliferation of MCF-7 human cancer Cells. These findings improve our understanding of the environmental transformation of EE2 in the shallow, anoxic waters.

Compost biofortification with diazotrophic and P-solubilizing bacteria improves maturation process and P availability.

BACKGROUND: Phosphorus-containing fertilizers play an important role in tropical agriculture owing to the well documented shortage of plant-available P in soils. Traditional P fertilizer production is based on chemical processing of insoluble rock phosphate (RP), which includes an acid treatment at high temperature. Processing the RP increases fertilizer costs, making it unavailable for undercapitalized and typically family-based farmers. Biotechnological methods have been proposed as an alternative to increase phosphate availability in RP. In this study, Burkholderia silvatlantica and Herbaspirillum seropedicae were co-inoculated into an RP-enriched compost with the aim of determining the effects of this technology on the levels of phosphatase activities and release of plant-available P. RESULTS: Inoculation of both microorganisms resulted in higher organic matter decomposition and higher humic acid formation in composting. Herbaspirillum seropedicae was the most promising microorganism for the production of acid and alkaline phosphatase enzymes. Both microorganisms presented potential to increase the supply of P from poorly soluble sources owing to increased levels of water-soluble P and citric acid P. CONCLUSION: Burkholderia silvatlantica and H. seropedicae in RP-enriched compost may represent an important biotechnological tool to reduce the overall time required for composting and increase the supply of P from poorly soluble sources. © 2016 Society of Chemical Industry.

Effects of multiple inhibitory components on anaerobic treatment processes in municipal solid waste incineration leachate.

This study served to investigate the comparative and combined effects of calcium, ammonia nitrogen, and aquatic humic substances (AHS) on specific methanogenic activity (SMA) in municipal solid waste leachate at mesophilic conditions. Using orthogonal experiments, anaerobic granular sludge was cultured with different concentrations combinations of the three added components for 13 days. The combination of 6000 mg/L calcium, 400 mg/L ammonia nitrogen, and 4000 mg/L AHS was the most inhibitory combination on the SMA of granular sludge, with a calculated 4.49 mL (standard temperature and atmospheric pressure) (STP) CH4/(gVSS·d) of SMA. The SMA with the addition of the inhibitory components was much lower than the control group's (1000 mg/L calcium, 200 mg/L ammonia nitrogen and 2000 mg/L AHS) with a calculated 12.97 mL (STP) CH4/(gVSS·d) of SMA. Calcium was the major inhibitor among the three components followed by AHS. High concentrations of calcium significantly inhibited the utilization of propionate and butyrate in the substrate and further affected the methanogenic process.

Rhizosphere processes are quantitatively important components of terrestrial carbon and nutrient cycles.

While there is an emerging view that roots and their associated microbes actively alter resource availability and soil organic matter (SOM) decomposition, the ecosystem consequences of such rhizosphere effects have rarely been quantified. Using a meta-analysis, we show that multiple indices of microbially mediated C and nitrogen (N) cycling, including SOM decomposition, are significantly enhanced in the rhizospheres of diverse vegetation types. Then, using a numerical model that combines rhizosphere effect sizes with fine root morphology and depth distributions, we show that root-accelerated mineralization and priming can account for up to one-third of the total C and N mineralized in temperate forest soils. Finally, using a stoichiometrically constrained microbial decomposition model, we show that these effects can be induced by relatively modest fluxes of root-derived C, on the order of 4% and 6% of gross and net primary production, respectively. Collectively, our results indicate that rhizosphere processes are a widespread, quantitatively important driver of SOM decomposition and nutrient release at the ecosystem scale, with potential consequences for global C stocks and vegetation feedbacks to climate.

The role of a novel fungal strain Trichoderma atroviride RVF3 in improving humic acid content in mature compost and vermicompost via ligninolytic and celluloxylanolytic activities.

AIMS: With the rising concerns about indiscriminate use of chemical fertilizers and accumulation of agro-industrial wastes in huge quantities, the present experiment was conducted to elucidate the effect of a novel fungal strain of Trichoderma atroviride in hastening the compost/vermicompost production process and for the production of humic acid (HA) rich compost and vermicompost. METHODS AND RESULTS: Rice (Oryza sativa) straw and distillation waste of geranium (Pelargonium graveolens), two important agricultural/industrial crop wastes were subjected to composting and vermicomposting. T. atroviride strains GVF10 (cellulase and xylanase producing), and RVF3 (ligninase and celluloxylanase producing) were inoculated alone or in combination. The HA content was found to increase maximally in rice vermicompost treated with ligninase producing strain RVF3 (85% with respect to HA standard) followed by geranium vermicompost + RVF3. The addition of the strain GVF10 increased HA content to about 35-62% with respect to HA standard. The addition of the fungal inoculum reduced the composting/vermicomposting time from 110 to 90 days. CONCLUSIONS: Our results indicate that the use of selected fungal strain(s) hold potential to produce qualitatively superior compost and vermicompost with high HA content in a shorter period. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of appropriate fungal strains may increase the efficiency of composting/vermicomposting processes producing compost and vermicompost with higher HA content, and alleviating the problems of solid waste accumulation and declining soil fertility.

Removal of humic acid from composted hog waste by the white-rot fungus, Phanerochaete chrysosporium.

The potential hazards of humic acid (HA) associated with hog waste effluent, coupled with increasing awareness of environmental problems, have prompted many countries to control disposal of effluents into water bodies and to maximize removal of HA. Here we employed the white-rot fungus, Phanerochaete chrysosporium, to degrade the HA in composted hog waste effluent, evaluated by the response surface method. Preliminary experiments demonstrate that pH, temperature and quantity of inoculum are significant variables determining success of the fungus. In total, 13 experiments were conducted with three variables designated as A (pH), B (temperature) and C (inoculum amount). The optimal conditions for reduction of HA by P. chrysosporium are pH 6, 31.5°C and an inoculum quantity of 5.86 g. Predicted and experimental results exhibit strong agreement, indicating efficiency in the model obtained by response surface method. Therefore, P. chrysosporium is an effective micro-organism for removal of HA from composted hog waste effluent.

A method suitable for DNA extraction from humus-rich soil.

A rapid and convenient method for extracting DNA from soil is presented. Soil DNA is extracted by direct cell lysis in the presence of EDTA, SDS, phenol, chloroform and isoamyl alcohol (3-methyl-1-butanol) followed by precipitation with 2-propanol. The extracted DNA is purified by modified DNA purification kit and DNA gel extraction kit. With this method, DNA extracted from humus-rich dark brown forest soil was free from humic substances and, therefore, could be used for efficient PCR amplification and restriction digestion. In contrast, DNA sample extracted with the traditional CTAB-based method had lower yield and purity, and no DNA could be extracted from the same soil sample with a commonly-used commercial soil DNA isolation kit. In addition, this method is time-saving and convenient, providing an efficient choice especially for DNA extraction from humus-rich soils.

A correlation between the fate and non-extractable residue formation of 14C-metalaxyl and enzymatic activities in soil.

Extracellular, oxidative soil enzymes like monophenol oxidases and peroxidases play an important role in transformation of xenobiotics and the formation of organic matter in soil. Additionally, these enzymes may be involved in the formation of non-extractable residues (NERs) of xenobiotics during humification processes. To examine this correlation, the fate of the fungicide (14)C metalaxyl in soil samples from Ultuna (Sweden) was studied. Using different soil sterilization techniques, it was possible to differentiate between free, immobilized, and abiotic ("pseudoenzyme"-like) oxidative activities. A correlation between the formation of metalaxyl NER and soil organic matter content, biotic activities, as well as extracellular phenoloxidase and peroxidase activities in the bulk soil and its particle size fractions was determined. Extracellular soil-bound enzymes were involved in NER formation (up to 8% of applied radioactivity after 92 days) of the fungicide independently from the presence of living microbes and different distributions of the NER in the soil humic subfractions.

[Transformation of humic substances of highly oxidized brown coal by the basidiomycetes fungi Trametes hirsuta and Trametes maxima].

The ability of the white rot basidiomycetes Trametes hirsuta and Trametes maxima to transform coal humic substances (HS's) under the conditions of solid phase cultivation in the presence or absence of an easily available source of corbon (glucose) has been studied. It was shown that during the growth of the fungal strains used in media, containing HS's, destructive and condensation processes of HS transformation proceeded simultaneously. Based on a comparative physicochemical analysis of the initial HS's and HS's transformed by the fungi, it was established that, despite the introduction of glucose may favor a deeper transformation of HS's by basidiomycetes, the general direction of their modification is dominant reduction or oxidation and is determined by the physiological biochemical peculiarities of the strain used.

How to enhance humification during composting of separately collected biowaste: impact of feedstock and processing.

Conventional parameters (loss on ignition, total organic carbon, total nitrogen, C/N-ratio, respiration activity (RA4), compost status (= 'Rottegrad'), NH4-N and NO3-N) are not correlated to humification. At best, they provide information on the biological stability (status of degradation) of composts. Humic substances which are a source of stable organic matter and nutrients are discussed as a parameter describing compost quality. Thus, in the present research project a photometric method evaluating humic acids was used to characterize the quality of 211 Austrian and foreign composts made from source-separated collected biowaste or sewage sludge. Furthermore, parameters influencing the formation of humic acids during the rotting process were investigated by implementing rotting experiments in the laboratory as well as in composting plants. The analysed composts showed humic acid contents between 2.5 and 47 %, calculated on a organic dry matter (oDM) basis. In addition to the duration of treatment the main influence on humification was the feedstock used. Stabilized sewage sludge, biowaste after intensive anaerobic pre-treatment or biowaste with low reactivity (RA4) or uniform composition (e.g. mainly grass) showed a low formation of humic acids. For optimum humification the feedstock needed to contain components that are well balanced from scarcely to easily degradable compounds. Processing also influenced humification. Open windrow systems and reactor systems allow the same quality to be produced when operated well, but optimizing mineralization (e.g. very intensive aeration) showed negative effects. The positive condition required for humification is an unhurried (not too intense) degradation with long-lasting biological activity in which microbes have enough time to use the metabolic products of degradation for humification.

[Earthworms as modifiers of the structure and biological activity of humic acids].

Passage of humic acids (HAs) through the digestive tract of the earthworm, Eiseniafetida andrei, resulted in a decrease in molecular masses of the HAs. The effect of earthworm-modified HAs on individual bacteria and on bacterial communities as a whole is different from the effect of native HAs. Modified HA probably induces and regulates microbial successions in soils and composts in a different manner than the native HA, suppressing or stimulating different groups of microorganisms. These results may explain why the positive effects of commercial humates in real soil ecosystems, unlike model communities, attenuate rapidly.

Involvement of laccase-like enzymes in humic substance degradation by diverse polar soil bacteria.

Humic substances (HS) in soil are widely distributed in cold environments and account for a significant fraction of soil's organic carbon. Bacterial strains (n = 281) were isolated at 15 °C using medium containing humic acids (HA), a principal component of HS, from a variety of polar soil samples: 217 from the Antarctic and 64 from the Arctic. We identified 73 potential HA-degrading bacteria based on 16S rRNA sequence similarity, and these sequences were affiliated with phyla Proteobacteria (73.9%), Actinobacteria (20.5%), and Bacteroidetes (5.5%). HA-degrading strains were further classified into the genera Pseudomonas (51 strains), Rhodococcus (10 strains), or others (12 strains). Most strains degraded HA between 10 and 25 °C, but not above 30 °C, indicating cold-adapted degradation. Thirty unique laccase-like multicopper oxidase (LMCO) gene fragments were PCR-amplified from 71% of the 73 HA-degrading bacterial strains, all of which included conserved copper-binding regions (CBR) I and II, both essential for laccase activity. Bacterial LMCO sequences differed from known fungal laccases; for example, a cysteine residue between CBR I and CBR II in fungal laccases was not detected in bacterial LMCOs. This suggests a bacterial biomarker role for LMCO to predict changes in HS-degradation rates in tundra regions as global climate changes. Computer-aided molecular modeling showed these LMCOs contain a highly-conserved copper-dependent active site formed by three histidine residues between CBR I and CBR II. Phylogenetic- and modeling-based methods confirmed the wide occurrence of LMCO genes in HA-degrading polar soil bacteria and linked their putative gene functions with initial HS-degradation processes.

Effects of Microbes from Coal-Related Commercial Humic Substances on Hydroponic Crop Cultivation: A Microbiological View for Agronomical Use of Humic Substances.

Here, coal-related humic substances (HSs) were examined to confirm whether sterilization treatments induce their inferior ability to stimulate lettuce in hydroponic cultivations. Interestingly, a drastic reduction in both lettuce biomass and microbial colony-forming units of the crop culture solutions was observed when the autoclaved HSs were treated. Some microbial genera (i.e., Bacillus and Aspergillus) identifiable in the bare HS-treated hydroponic systems were able to be isolated by direct inoculation of bare HS powders on conventional microbial nutrients, supporting that flourishing microbes in the hydroponic cultivations derive from bare HSs-treated. Moreover, coincubation of some isolated bacterial and fungal strains (i.e., Bacillus and Aspergillus genera) from HSs with lettuce resulted in a significant increase in plant biomass and enhanced resistance to NaCl-related abiotic stresses. Microbial volatile organic compounds renowned for plant stimulation were detected by using solid-phase microextraction coupled with gas chromatography-mass spectrometry. It was finally confirmed that the isolates are capable of utilizing carbon substrates such as pectin and tween 20 or 40, which are relevant to those of microbes isolated from peat and leonardite (i.e., HS extraction sources). Overall, our results suggest that microbiological factors could be considered when commercial coal-related HSs are applied in hydroponic crop cultivations.

Effects of extracellular polymeric and humic substances on chlorpyrifos bioavailability to Chironomus riparius.

The role of sediment organic matter quality and quantity for chlorpyrifos bioavailability was studied in experiments with Chironomus riparius larvae and with four types of organic matter; (1) commercially available extracellular polymeric substances (EPS), (2) EPS produced by sediment microbes, (3) commercially available humic substances and, (4) humic substances extracted from a boreal lake. The effects of each type of organic matter were assessed at three concentrations. We used a (14)C-tracer approach to quantify uptake of chlorpyrifos in the larvae, and the partitioning of the insecticide within the microcosm. Carbon-normalised larval uptake was reduced both by EPS and humic substances. However, the reduction in uptake was much greater for EPS than for humic substances: uptake was reduced by 94 and 88% for commercial and complex EPS, and by 59 and 57% for commercial and complex humic substances, respectively. We also found differences in chlorpyrifos uptake, and sediment concentrations between treatments with commercially available and complex polymers, suggesting that minor differences in the quality of relatively simple organic molecules can affect contaminant behaviour in ecotoxicological studies. Passive uptake in dead controls was 40% of that in living larvae. Therefore, both passive and digestive uptake were important processes for chlorpyrifos uptake by larvae. Our results show that both EPS and humic substances affect chlorpyrifos bioavailability to sediment biota negatively and contribute to the understanding of the processes that regulate organic contaminant bioavailability in aquatic environments.

Recovery of efficient treatment performance in a semi-aerobic aged refuse biofilter when treating landfill leachate: Washing action using domestic sewage.

Semi-aerobic aged refuse biofilters (SAARB) are known to efficiently remove organic matter, nitrogenous substances, and anions from landfill leachate. However, long-term recirculation of mature landfill leachate inevitably leads to accumulation of pollutants and decreases treatment capacity. In this study, the washing action provided by domestic sewage was used to recover and even enhance the treatment performance of SAARBs treating mature landfill leachate. Three SAARB columns were operated for 300 d after which a "Recirculation-Washing-Recirculation" sequence was followed. In the first recirculation period (22 d), removal of chemical oxygen demand (COD) and total nitrogen (TN) decreased from ca. 90% and 60%, respectively, initially to about 75% and less than 20%, respectively. Thereafter, washing (20 d) of the SAARBs was accomplished by applying domestic sewage. In the subsequent second recirculation period (30 d), the SAARBs were operated at the same hydraulic loading as used initially, but achieved high (ca. 90%) COD and relatively high (ca. 59%-76%) TN removal, including degradation of refractory organic matter such as humic- and fulvic-like substances. Overall, the mechanisms of the treatment performance recovery (including organics degradation and nitrification-denitrification) using domestic sewage can be attributed to three main effects: (1) some accumulated pollutants were washed out, thereby leading to recovery of the adsorption ability of aged refuse; (2) the inhibition of bio-refractory organics stress on microbial activities was mitigated by domestic sewage washing; and (3) the wash out of some accumulated salts (e.g., chloride and sulfate ions) probably helped the microbial activity recover.