Ronald R. Navarro

Bacterial population succession and adaptation affected by insecticide application and soil spraying history

Although microbial communities have varying degrees of exposure to environmental stresses such as chemical pollution, little is known on how these communities respond to environmental disturbances and how past disturbance history affects these community-level responses. To comprehensively understand the effect of organophosphorus insecticide application on microbiota in soils with or without insecticide-spraying history, we investigated the microbial succession in response to the addition of fenitrothion [O,O-dimethyl O-(3-methyl-p-nitrophenyl) phosphorothioate, abbreviated as MEP] by culture-dependent experiments and deep sequencing of 16S rRNA genes. Despite similar microbial composition at the initial stage, microbial response to MEP application was remarkably different between soils with and without MEP-spraying history. MEP-degrading microbes more rapidly increased in the soils with MEP-spraying history, suggesting that MEP-degrading bacteria might already exist at a certain level and could quickly respond to MEP re-treatment in the soil. Culture-dependent and -independent evaluations revealed that MEP-degrading Burkholderia bacteria are predominant in soils after MEP application, limited members of which might play a pivotal role in MEP-degradation in soils. Notably, deep sequencing also revealed that some methylotrophs dramatically increased after MEP application, strongly suggesting that these bacteria play a role in the consumption and removal of methanol, a harmful derivative from MEP-degradation, for better growth of MEP-degrading bacteria. This comprehensive study demonstrated the succession and adaptation processes of microbial communities under MEP application, which were critically affected by past experience of insecticide-spraying.

Evidence of Environmental and Vertical Transmission of Burkholderia Symbionts in the Oriental Chinch Bug, Cavelerius saccharivorus (Heteroptera: Blissidae)

ABSTRACT The vertical transmission of symbiotic microorganisms is omnipresent in insects, while the evolutionary process remains totally unclear. The oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae), is a serious sugarcane pest, in which symbiotic bacteria densely populate the lumen of the numerous tubule-like midgut crypts that the chinch bug develops. Cloning and sequence analyses of the 16S rRNA genes revealed that the crypts were dominated by a specific group of bacteria belonging to the genus Burkholderia of the Betaproteobacteria. The Burkholderia sequences were distributed into three distinct clades: the Burkholderia cepacia complex (BCC), the plant-associated beneficial and environmental (PBE) group, and the stinkbug-associated beneficial and environmental group (SBE). Diagnostic PCR revealed that only one of the three groups of Burkholderia was present in ∼89% of the chinch bug field populations tested, while infections with multiple Burkholderia groups within one insect were observed in only ∼10%. Deep sequencing of the 16S rRNA gene confirmed that the Burkholderia bacteria specifically colonized the crypts and were dominated by one of three Burkholderia groups. The lack of phylogenetic congruence between the symbiont and the host population strongly suggested host-symbiont promiscuity, which is probably caused by environmental acquisition of the symbionts by some hosts. Meanwhile, inspections of eggs and hatchlings by diagnostic PCR and egg surface sterilization demonstrated that almost 30% of the hatchlings vertically acquire symbiotic Burkholderia via symbiont-contaminated egg surfaces. The mixed strategy of symbiont transmission found in the oriental chinch bug might be an intermediate stage in evolution from environmental acquisition to strict vertical transmission in insects.

Application of aqueous saponin on the remediation of polycyclic aromatic hydrocarbons-contaminated soil

The aim of this research was to evaluate the feasibility of aqueous saponin for the removal and biodegradation of polycyclic aromatic hydrocarbons (PAHs) from contaminated soil. Dissolution test confirmed the ability of saponin to increase the apparent solubility of the tested 3–5 rings PAH above the critical micelle concentration (approximately 1000 mg/L). Microbial test with pure culture of Sphingomonas sp. showed that saponin significantly enhanced the degradation of pyrene. For example, the percent degradation was 2.1 times higher in the presence of 2500 mg/L saponin than that of control without saponin after 60 hours incubation at around 108 CFU/mL initial cell loading. These results suggest that the binding of pyrene with saponin does not pose a serious constraint to bacterial uptake. Contrary to pyrene, saponin was chemically stable against the PAHs degrader. It is also not toxic to the cell at least up to 2500 mg/L. Finally, using a spiked soil sample, extraction tests with 10,000 mg/L of saponin showed that around 52.7% and 0.3% of pyrene was removed from low and high organic spiked soils, respectively. The results from this study indicate that aqueous saponin is appropriate as a washing agent as well as biodegradation enhancer for the detoxification of PAHs-contaminated low organic carbon soil.

Treatment of PAHs in contaminated soil by extraction with aqueous DNA followed by biodegradation with a pure culture of Sphingomonas sp

The biodegradation of polycyclic aromatic hydrocarbons (PAHs) in aqueous deoxyribonucleic acid (DNA) solution from contaminated soil washing was investigated. Initial data with a model effluent consisting of anthracene, phenanthrene, pyrene and benzo[a]pyrene that were individually dissolved in 1% aqueous DNA solution confirmed their positive degradation by Sphingomonas sp. at around 10(8)CFU mL(-1) initial cell loading. For anthracene and phenanthrene, complete removal was achieved within 1h treatment. Degradation of pyrene and benzo[a]pyrene took a relatively longer time of a few days and weeks, respectively. DNA-dissolved PAHs were also degraded relatively faster than PAH crystals in aqueous medium to suggest that the binding of the PAHs in the polymer does not pose serious constraint to bacterial uptake. The DNA was stable against the PAH-degrading bacteria. Parallel experiments with actual DNA solutions obtained during pyrene extraction from an artificially spiked soil also showed similar results. Close to 100% pyrene degradation was achieved after 1d treatment. With its chemical stability, the cell-treated DNA was re-used up to four cycles without a considerable decline in extraction performance.

High-resolution phylogenetic analysis of residual bacterial species of fouled membranes after NaOCl cleaning.

Biofouling is one of the major problems during wastewater treatment using membrane bioreactors (MBRs). In this regard, sodium hypochlorite (NaOCl) has been widely used to wash fouled membranes for maintenance and recovery purposes. Advanced chemical and biological characterization was conducted in this work to evaluate the performance of aqueous NaOCl solutions during washing of polyacrylonitrile membranes. Fouled membranes from MBR operations supplemented with artificial wastewater were washed with 0.1% and 0.5% aqueous NaOCl solutions for 5, 10 and 30 min. The changes in organics composition on the membrane surface were directly monitored by an attenuated total reflection Fourier transform infrared (ATR-FT-IR) spectrometer. In addition, high-throughput Illumina sequencing of 16S rRNA genes was applied to detect any residual microorganisms. Results from ATR-FT-IR analysis indicated the complete disappearance of functional groups representing different fouling compounds after at least 30 min of treatment with 0.1% NaOCl. However, the biomolecular survey revealed the presence of residual bacteria even after 30 min of treatment with 0.5% NaOCl solution. Evaluation of microbial diversity of treated samples using Chao1, Shannon and Simpson reciprocal indices showed an increase in evenness while no significant decline in richness was observed. These implied that only the population of dominant species was mainly affected. The high-resolution phylogenetic analysis revealed the presence of numerous operational taxonomic units (OTUs) whose close relatives exhibit halotolerance. Some OTUs related to thermophilic and acid-resistant strains were also identified. Finally, the taxonomic analysis of recycled membranes that were previously washed with NaOCl also showed the presence of numerous halotolerant-related OTUs in the early stage of fouling. This further suggested the possible contribution of such chemical tolerance on their survival against NaOCl washing, which in turn affected their re-fouling potential.

High-Resolution Dynamics of Microbial Communities during Dissimilatory Selenate Reduction in Anoxic Soil

Selenate is one of the most common toxic metal compounds in contaminated soils. Its redox status can be changed by microbial activity, thus affecting its water solubility and soil mobility. However, current knowledge of microbial dynamics has been limited by the low sensitivity of past isolation and identification protocols. Here, high-throughput Illumina sequencing of 16S rRNA genes was applied to monitor the shift of the microorganisms in an anoxic contaminated soil after Se(VI) and acetate amendment. An autoclaved soil with both chemicals and a live soil with acetate alone were used as controls. Preliminary chemical analysis clearly showed the occurrence of biological selenate reduction coupled with acetate oxidation. Principal coordinate analysis and diversity indices of Illumina-derived sequence data showed dynamic succession and diversification of the microbial community in response to selenate reduction. High-resolution phylogenetic analysis revealed that the relative frequency of an operational taxonomic unit (OTU) from the genus Dechloromonas increased remarkably from 0.2% to 36% as a result of Se(VI) addition. Multiple OTUs representing less abundant microorganisms from the Rhodocyclaceae and Comamonadaceae families had significant increases as well. This study demonstrated that these microorganisms are concertedly involved in selenate reduction of the employed contaminated soil under anoxic conditions.

Influence of compost amendment on pyrene availability from artificially spiked soil to two subspecies of Cucurbita pepo

The dissolved organic matter (DOM) fraction of soil organic matter (SOM) may positively contribute to polycyclic aromatic hydrocarbons (PAHs) bioavailability. This work investigated the effects of DOM-rich and PAHs-free compost amendment on the plant uptake of pyrene. Two subspecies of Cucurbita pepo (ssp. pepo cv. Raven and ssp. texana cv. Sunray) were grown for three weeks in a spiked soil containing 83.9 mg kg(-1) pyrene under four different treatments; inorganic fertilizer (IF) alone, 15% (v/v) mixed gardening compost with IF (MX15%+IF), MX30% alone, and no fertilization (NF). Equilibrium pyrene desorptions from a spiked soil (104 mg kg(-1)) under different concentrations (35-590 mg-C L(-1)) of DOM extracts derived from two types of composts including MX and cow manure were also conducted. After harvest, the decrease in the pyrene concentration of the soil ranged from 46-65% for the different treatments. The total dry biomass for both plants was highest under MX15%+IF. The bioconcentration factors of pyrene for both also tended to decrease with increasing MX dose from 15% to 30%. However, the total uptakes of pyrene with IF and MX15%+IF were not statistically different (36.7 and 33.7 microg for Raven, and 5.20 and 7.90 microg for Sunray, respectively). These values were around 100% higher than that with NF (17.4 microg for Raven and 2.0 microg for Sunray). The pyrene desorption data confirmed the ability of DOM to associate with pyrene as indicated by its increase in apparent water solubility. On the basis of these results, MX application at 15% (v/v) does not significantly reduce the phytoextraction of pyrene due to the enhancement of plant growth as well as the possible contribution of DOM fractions to pyrene bioavailability. The application of compost may not pose serious concerns regarding the efficiency of phytoremediation of PAHs-polluted soil.

Fine-scale monitoring of shifts in microbial community composition after high organic loading in a pilot-scale membrane bioreactor.

In biological wastewater treatment, municipal wastewater sometimes undergoes unexpected changes in physicochemical parameters, such as organic carbon concentration. The aim of this study was to understand how microbial communities in activated sludge in a membrane bioreactor (MBR) adapt to high organic loading and maintain their degradation ability during reactor operation. A pilot-scale MBR was operated for 19 days. On day 8, the concentration of organic matter in the synthetic wastewater increased from 450 to 900 mg chemical oxygen demand (COD)/L. Even under conditions of high organic loading, COD removal rates were high, ranging from 85.3 to 91.4%. High-throughput sequencing of 16S rRNA genes revealed that microbial communities changed drastically with increased organic loading. After day 8, Aquabacterium- and Azospira-related operational taxonomic units (OTUs) belonging to the class β-proteobacteria became dominant; this potentially enhanced the degradation of organic substances and decreased activated sludge microbial diversity. Due to the use of dissolved oxygen (DO) for degradation of organic substances, DO levels in the reactor decreased. This led to an increase in a subset of OTUs related to not only aerobic but also anaerobic bacteria, e.g., those in the class Clostridia. During this period, anaerobic microorganisms may have contributed to the degradation of organic substances to maintain MBR performance. On the other hand, high-throughput sequencing also made it possible to identify yet-to-be cultured or minor microorganisms affiliated with the candidate phylogenetic division SR1 and ammonia-oxidizing archaea in activated sludge.

Ferrite formation from photo-Fenton treated wastewater.

Photo-Fenton oxidation followed by ferrite formation was applied for the degradation of a representative organic compound, phenol, and the subsequent removal of the Fe ions. At a phenol:Fe(II):H(2)O(2) molar ratio of 1:0.5:15, TOC analysis showed almost complete mineralization of 10.6mM phenol after 2h at a controlled pH of 3. Recalcitrant low molecular weight organic acid by-products particularly oxalic acid were destroyed. A ferrous-rich solution was generated so that alkalinization at pH 10.5 generated a pitch black sludge of lower volume and moisture content than a ferric hydroxide control of the same Fe concentration. The flocs exhibited a strong affinity for a magnet and its X-ray diffraction pattern showed a close similarity to a standard spinel magnetite. With proper monitoring of Fe(II) and dissolved oxygen, the reaction was successfully controlled to generate flocs with more than 30% magnetite content. When photo-Fenton was employed as a pre-treatment step so that residual oxalic acid remained, ferrite formation was not inhibited. The presence of oxalates even allowed ferrites to form in a solution containing Ca(2+) ions, which is well-known to be deterrent to the process.

Enhancement of phosphonomethylated PEI–Cu2+complex flocculation by Ca2+ ions: A new approach for heavy metal removal from aqueous solutions

Removal of Cu 2+ ions from aqueous solutions by induced flocculation of PPEI with Ca 2+ ions was studied. Considerable floc formation accompanying Cu 2+ sequestration was demonstrated even at low initial metal concentration of 5 mg L− 1. Flocculation capacity as high as 4.7 mmol Cu 2+/g PPEI at pH 6 was obtained. Ca 2+ showed minimal competition with Cu 2+ ions for adsorption sites in the polymer. The PPEI–Ca 2+ flocculant system was effective over a wide range of pH so that at an equilibrium pH of 1.5, more than 50% Cu 2+ removal was possible. The [CaCl2]/[PPEI] ratio is a significant parameter with regards to achieving complete Cu 2+ removal. At optimized condition, the process is capable of scavenging Cu 2+ ions to background levels.