Edible insects: Understanding benzo(a)pyrene toxicokinetics in yellow mealworms for safe and sustainable consumption.

The global interest in edible insects as sustainable protein sources raises concerns about the bioaccumulation of contaminants, including polycyclic aromatic hydrocarbons (PAHs), to problematic levels. Understanding the accumulation dynamics of PAHs in edible insects is highly relevant due to the widespread sources and toxicological profiles; however, the bioaccumulative potential of PAHs in edible insects is unexplored. This study examined the uptake and elimination dynamics of benzo(a)pyrene (B(a)P), a representative and carcinogenic PAH, in yellow mealworm larvae (YMW, Tenebrio molitor). Larvae were exposed to feeding substrate with varying B(a)P concentrations (0.03, 0.3, and 3 mg kg-1), and uptake (21 days in B(a)P-contaminated substrate) and elimination (21 days in B(a)P-free substrate) kinetics were subsequently assessed. The results showed that YMW can eliminate B(a)P, revealing dose-dependent B(a)P bioaccumulation in these insects. Larvae fed on a substrate with 0.03 mg kg-1 accumulated B(a)P over 21 days, presenting values of 0.049 (Standard deviation - 0.011) mg kg-1 and a kinetic-based (BAFkinetic) of 1.93 g substrate g organism-1, exceeding the EU regulatory limits for food. However, with a B(a)P half-life (DT50) of 4.19 days in the larvae, an EU legislation safety criterion was met after a 13-day depuration period in clean substrate. Larvae exposed to substrates with 0.3 and 3 mg kg-1 showed B(a)P accumulation, with BAFkinetic values of 3.27 and 2.09 g substrate g organism-1, respectively, not meeting the current legal standards for food consumption at the end of the exposure to B(a)P. Although the B(a)P half-life values after 35 days were 4.30 and 10.22 days (DT50s), the larvae retained B(a)P levels exceeding permitted food safety limits. These findings highlight a significant oversight in regulating PAHs in animal feed and the need for comprehensive safety evaluations of PAH hazards in edible insects for improved PAH feeding guidelines.

Simultaneous biofiltration of H2S, NH3, and toluene using compost made of chicken manure and sugarcane bagasse as packing material.

Offensive odors from wastewater treatment plants (WWTP) are caused by volatile inorganic compounds such as hydrogen sulfide and ammonia and volatile organic compounds (VOCs), such as toluene. To treat these pollutants, biofiltration is an effective and economical technology used worldwide due to its low investment and environmental impact. In this work, a laboratory-scale prototype biofilter unit for the simultaneous biofiltration of hydrogen sulfide, ammonia, and toluene was evaluated by simulating the emission concentrations of the El Salitre WWTP Bogotá, Colombia, using a compost of chicken manure and sugarcane bagasse as packing material for the biofilter. The prototype biofilter unit was set to an operation flow rate of 0.089 m3/h, an empty bed residence time (EBRT) of 60 s, and a volume of 0.007 m3 (6.6 L). The maximum removal efficiency were 96.9 ± 1.2% for H2S, at a loading rate of 4.7 g/m3 h and a concentration of 79.1 mg/m3, 68 ± 2% for NH3, at a loading rate of 1.2 g/m3 h and a concentration of 2.0 mg/m3, and 71.5 ± 4.0% for toluene, at a loading rate of 1.32 g/m3 h and a concentration of 2.3 mg/m3. The removal efficiency of the three compounds decreased when the toluene concentration was increased above 40 mg/m3. However, a recovery of the system was observed after reducing the toluene concentration and after 7 days of inactivity, indicating an inhibitory effect of toluene. These results demonstrate the potential use of the prototype biofilter unit for odor treatment in a WWTP.

Potential use of two Serratia strains for cadmium remediation based on microbiologically induced carbonate precipitation and their cadmium resistance.

Cadmium (Cd) presence and bioavailability in soils is a serious concern for cocoa producers. Cocoa plants can bioaccumulate Cd that can reach humans through the food chain, thus posing a threat to human health, as Cd is a highly toxic metal. Currently, microbiologically induced carbonate precipitation (MICP) by the ureolytic path has been proposed as an effective technique for Cd remediation. In this work, the Cd remediation potential and Cd resistance of two ureolytic bacteria, Serratia sp. strains 4.1a and 5b, were evaluated. The growth of both Serratia strains was inhibited at 4 mM Cd(II) in the culture medium, which is far higher than the Cd content that can be found in the soils targeted for remediation. Regarding removal efficiency, for an initial concentration of 0.15 mM Cd(II) in liquid medium, the maximum removal percentages for Serratia sp. 4.1.a and 5b were 99.3% and 99.57%, respectively. Their precipitates produced during Cd removal were identified as calcite by X-ray diffraction. Energy dispersive X-ray spectroscopy analysis showed that a portion of Cd was immobilized in this matrix. Finally, the presence of a partial gene from the czc operon, involved in Cd resistance, was observed in Serratia sp. 5b. The expression of this gene was found to be unaffected by the presence of Cd(II), and upregulated in the presence of urea. This work is one of the few to report the use of bacterial strains of the Serratia genus for Cd remediation by MICP, and apparently the first one to report differential expression of a Cd resistance gene due to the presence of urea.

Multidimensional chromatography in environmental analysis: Comprehensive two-dimensional liquid versus gas chromatography.

Analysis of complex environmental matrices poses an extreme challenge for analytical chemists due to the vast number of known and unknown compounds, with very diverse chemical and physical properties. The need for a holistic characterisation of this complexity has sparked the development of effective tools to unravel the chemical composition of such environmental samples. Multidimensional chromatographic methods, namely comprehensive two-dimensional (2D) gas and liquid chromatography (GC × GC and LC × LC, respectively), coupled to different detection systems have emerged as powerful tools with the capability to address this challenge. While GC × GC has steadily gained popularity in environmental analysis, LC × LC is surprisingly less attractive in this research field. This critical review article explores the potential reasons why LC × LC is not the dominant technique used in environmental analysis as compared to GC × GC, while simultaneously highlighting the quite unique role of LC × LC for the target and untargeted analysis of complex environmental matrices. The possible combinations of stationary phases, the important role of the interfacing valve as the heart of an LC × LC assembly, the existing optimization strategies for improving the separation power in the 2D chromatographic space, and the need for user-friendly mathematical tools for multidimensional data handling are also discussed. Finally, a set of practical measures are suggested to increase the use and secure the success of LC × LC in environmental analysis.

Isolation of urease-producing bacteria from cocoa farms soils in Santander, Colombia, for cadmium remediation.

Cadmium (Cd) is a toxic heavy metal that causes serious health problems and is present in agriculturally important soils in Colombia, such as the ones used for cocoa farming. Recently, the use of ureolytic bacteria by the Microbiologically Induced Carbonate Precipitation (MICP) activity has been proposed as an alternative to mitigate the availability of Cd in contaminated soils. In this study, 12 urease-positive bacteria able to grow in the presence of Cd(II) were isolated and identified. Three were selected based on urease activity, precipitates formation and growth, with two belonging to the genus Serratia (codes 4.1a and 5b) and one to Acinetobacter (code 6a). These isolates exhibited low urease activity levels (3.09, 1.34 and 0.31 µmol mL-1 h-1, respectively), but could raise the pH to values close to 9.0 and to produce carbonate precipitates. It was shown that the presence of Cd affects the growth of the selected isolates. However, urease activity was not negatively influenced. In addition, the three isolates were observed to efficiently remove Cd from solution. The two Serratia isolates presented maximum removals of 99.70% and 99.62%, with initial 0.05 mM Cd(II) in the culture medium (supplemented with urea and Ca(II)) at 30 °C and 144 h of incubation. For the Acinetobacter isolate, the maximum removal was 91.23% at the same conditions. Thus, this study evidences the potential use of these bacteria for bioremediation treatments in samples contaminated with Cd, and it is one of the few reports that shows the high cadmium removal capacity of bacteria from the genus Serratia. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03495-1.

Use of Trichoderma koningiopsis chitinase to enhance the insecticidal activity of Beauveria bassiana against Diatraea saccharalis.

Trichoderma is a well-known soil-borne fungus, highly efficient producer of extracellular enzymes including chitinases. The aim of this study was to recover a chitinase from fermentation waste after harvesting Trichoderma koningiopsis Th003 conidia and assess its potential as an enhancer of Beauveria bassiana insecticidal activity against Diatraea saccharalis. T. koningiopsis was produced by solid fermentation, conidia were harvested, and a crude extract (CE) was recovered by washing the residual substrate (rice:wheat bran). The partially purified chitinase (PPC) (75 kDa product) with N-acetyl-ß-glucosaminidase activity was obtained by chromatography to 29.3-fold with optimal activity at pH 5 and 55°C. Both the CE and the PPC were mixed with B. bassiana Bv062 conidia and assessed in a bioassay against D. saccharalis larvae. The CE and PPC from T. koningiopsis Th003 did not affect the germination or viability of B. bassiana conidia and enhanced its insecticidal activity when used at 0.06 U/ml enzymatic activity with a 24.5% reduction in B. bassiana lethal time (LT90 ). This study demonstrated the potential of chitinases produced by T. koningiopsis in solid fermentation to be recovered from the waste substrate and used as an additive to enhance B. bassiana, adding value to the main waste from the Trichoderma biopesticide/biofertilizer industries.

AISLAMIENTO E IDENTIFICACIÓN DE Lactobacillus spp. (LACTOBACILLACEAE) RESISTENTES A Cd(II) Y As(III) RECUPERADOS DE FERMENTO DE CACAO / Isolation and identification of Lactobacillus spp. (Lactobacillaceae) resistant to Cd(II) and As(III) from fermented cocoa

RESUMEN El objetivo de este estudio fue aislar e identificar a partir de cacao fermentado en Caldas Colombia, bacterias con potencial de aplicación en procesos biotecnológicos, como la detoxificación de cadmio (Cd(II)) y arsénico (As(III)) en el organismo humano. En total se recuperaron 36 aislados de los cuales se recuperaron 11 en presencia de 1,0 mg/L de Cd(II) y 25 en presencia de 0,1 mg/L de As(III). Su identificación molecular determinó que la mayoría de los aislados son del género Lactobacillus. Los ensayos de crecimiento en presencia de diferentes concentraciones de los elementos evaluados permitió determinar que gran parte de los aislamientos presentan resistencia a mayores concentraciones de As(III) (300 mg/L) que de Cd(II) (10 mg/L). En ensayos de tolerancia a la acidez (pH 2,5) se encontró que la cepa tipo Lactobacillus plantarum JCM 1055, junto con los aislamientos nativos L. plantarum A19, A26 y C16, mostraron la mayor tolerancia, por lo que se seleccionaron para evaluar su tolerancia a condiciones de salinidad. Las bacterias evaluadas mostraron crecimiento en concentraciones de hasta 4 g/L de sales biliares. Se concluye que los L. plantarum evaluados en este trabajo tienen un gran potencial para futuros ensayos en los que se busque demostrar la disminución de la bioaccesibilidad de Cd(II) y As(III) en condiciones in vitro del sistema digestivo humano debido a su resistencia a altas concentraciones de estos elementos y su tolerancia a condiciones de acidez y salinidad. Esto, junto con el reconocido potencial probiótico que tienen estos microorganismos, permitirá a futuro su uso en procesos biológicos de mitigación de Cd(II) y As(III).

ABSTRACT The aim of this study was to isolate and identify from fermented cocoa in Caldas Colombia, bacteria with potential application in biotechnological processes such as detoxification of cadmium (Cd(II)) and arsenic (As(III)) in the human organism. In total, 36 isolates were obtained, from which 11 were recovered in the presence of 1.0 mg/L of Cd(II) and 25 in presence of 0.1 mg/L of As(III). Molecular identification showed most isolates belong to the genera Lactobacillus. Minimum inhibitory concentration assays, in presence of different concentrations of the elements, allowed to determine that the majority of isolates have resistance to higher concentration of As(III) (300 mg/L) than Cd(II) (10 mg/L). Acidity tolerance assays at pH 2.5 showed that type strain Lactobacillus plantarum JCM 1055, and native isolates L. plantarum A19, A26, and C16, presented the highest tolerance, thus they were selected to evaluate their tolerance to salinity conditions. The evaluated bacteria could grow in bile salts up to 4 g/L. It is concluded that the evaluated L. plantarum have great potential to be used in assays in which bioaccessibility of Cd(II) and As(III) is diminished under in vitro conditions of the human digestive system, due to its resistance to high concentrations of the elements and tolerance to acidic and high bile salt conditions. These facts, together with the recognized probiotic potential of these microorganisms, may allow their future use in biological processes to mitigate Cd(II) and As(III).

Simultaneous biofiltration of H2S and NH3 using compost mixtures from lignocellulosic waste and chicken manure as packing material.

Biofiltration offers an efficient and economical alternative for the elimination of offensive odors caused by hydrogen sulfide, ammonia, and volatile organic compounds. Considering that packing materials affect the performance and represent the main installation cost, the purpose of this work was to evaluate the biofiltration of H2S and NH3 comparing three composted mixtures made from chicken manure and lignocellulosic residues (pruning waste, sugarcane bagasse, and rice husk) used as packing material. A range of gas concentrations similar to those of a municipal WWTP was used in the biofiltration of a contaminated stream performed on a laboratory scale. The results indicate that at low concentrations of H2S (6-36 ppm) and NH3 (0-1 ppm), the three biofilters showed 100% removal efficiency. Now, at the maximum levels of gas concentrations of H2S (250 ppm) and NH3 (19 ppm) while the removal efficiency of H2S remained higher than 90% in all cases, the removal efficiency of NH3 remained higher than 90% only in the sugarcane bagasse biofilter. Compost mixtures with sugarcane bagasse and rice husk are highly reliable as packing material for biofiltration at high concentration of H2S. Specifically, the sugarcane bagasse mixture had the highest removal efficiency (99% H2S and 95% NH3) and the highest elimination capacity (15 g H2S/m3h and 0.6 g NH3/m3h), making it a better option for the elimination of both gases. These results represent a contribution to the construction of a low-price elimination system of offensive odors in WTTPs and other industries.

Metal and metalloid immobilization by microbiologically induced carbonates precipitation.

The industrialization and growth of human population has increased the release and accumulation of metals and metalloids in the environment. Bioaccumulation and exposure to these elements have been associated with different types of diseases and cancer, thus looking for alternatives that decrease their bioavailability in the environment is crucial. Microbiologically induced carbonates precipitation (MICP) has been proposed as a potential bioremediation method to immobilize contaminating metals and metalloids. Studies published to date have mainly used ureolytic bacteria, reporting metal(loid)s removal percentages up to 100% for some toxic elements, thus demonstrating the effectiveness of this treatment. Various genera of bacteria, particularly Gram-positive, have been reported with MICP abilities. More recently, fungi have also been proposed as a viable alternative for the removal of these toxic elements by carbonate precipitation. This mini-review presents updated information about the main studies carried out to date using different types of microorganisms that perform MICP to decrease the environmental bioavailability of toxic metals and metalloids through the formation of metallic carbonates.

Stable and Enriched Cenarchaeum symbiosum and Uncultured Betaproteobacteria HF1 in the Microbiome of the Mediterranean Sponge Haliclona fulva (Demospongiae: Haplosclerida).

Sponges harbor characteristic microbiomes derived from symbiotic relationships shaping their lifestyle and survival. Haliclona fulva is encrusting marine sponge species dwelling in coralligenous accretions or semidark caves of the Mediterranean Sea and the near Atlantic Ocean. In this work, we characterized the abundance and core microbial community composition found in specimens of H. fulva by means of electron microscopy and 16S amplicon Illumina sequencing. We provide evidence of its low microbial abundance (LMA) nature. We found that the H. fulva core microbiome is dominated by sequences belonging to the orders Nitrosomonadales and Cenarchaeales. Seventy percent of the reads assigned to these phylotypes grouped in a very small number of high-frequency operational taxonomic units, representing niche-specific species Cenarchaeum symbiosum and uncultured Betaproteobacteria HF1, a new eubacterial ribotype variant found in H. fulva. The microbial composition of H. fulva is quite distinct from those reported in sponge species of the same Haliclona genus. We also detected evidence of an excretion/capturing loop between these abundant microorganisms and planktonic microbes by analyzing shifts in seawater planktonic microbial content exposed to healthy sponge specimens maintained in aquaria. Our results suggest that horizontal transmission is very likely the main mechanism for symbionts' acquisition by H. fulva. So far, this is the first shallow water sponge species harboring such a specific and predominant assemblage composed of these eubacterial and archaeal ribotypes. Our data suggests that this symbiotic relationship is very stable over time, indicating that the identified core microbial symbionts may play key roles in the holobiont functioning.

4-hydrazinobenzoic acid as a derivatizing agent for aldehyde analysis by HPLC-UV and CE-DAD.

Aldehydes are relevant analytes in a wide range of samples, in particular, food and beverages but also body fluids. Hydrazines can undergo nucleophilic addition with aldehydes or ketones giving origin to hydrazones (a group of stable imines) that can be suitably used in the identification of aldehydes. Herein, 4-hydrazinobenzoic acid (HBA) was, for the first time, used as the derivatizing agent in analytical methodologies using liquid chromatography aiming the determination of low-molecular aldehydes. The derivatization reaction was simultaneously performed along with the extraction process, using gas-diffusion microextraction (GDME), which resulted in a clean extract containing the HBA-aldehyde derivates. The corresponding formed imines were determined by both high-performance liquid chromatography (LC) with UV spectrophotometric detection (HPLC-UV) and capillary electrophoresis with diode array detection (CE-DAD). HBA showed to be a rather advantageous derivatization reagent due to its stability, relatively high solubility in water and other solvents, high selectivity and sensibility, reduced impurities, simple preparation steps and applicability to different separation and/or different detection techniques. Limits of detections (LODs) of the optimized methodologies (in terms of time and pH among other experimental variables) were all below 0.5 mg L-1, using both instrumental techniques. Furthermore, for the first time, the HBA-aldehyde derivatives were analyzed by LC with mass spectrometry (LC-MS), demonstrating the possibility of identification by MS of each compound. The developed methodologies were also successfully applied in the analysis of formaldehyde and acetaldehyde in several alcoholic beverages. This was also the first time GDME was combined with CE, showing that it can be a valuable sample preparation tool for electrophoresis, in particular by eliminating the interference of ions and inorganic constituents present in the samples.

Isolation and Potential Biocementation of Calcite Precipitation Inducing Bacteria from Colombian Buildings.

Microbiological induced calcium carbonate or calcite precipitation (MICP) has become a highly researched issue due to its multiple applications in the construction industry, being a promising alternative with a great biotechnological importance. In this work, potential calcite precipitation inducing bacteria were isolated from mortar and concrete samples of different buildings at the National University of Colombia. Eighteen crystal-precipitating strains were recovered in Urea-CaCl2 solid medium. The 16S rRNA gene sequencing identified isolates as Arthrobacter, Psychrobacillus and Rhodococcus genera. It is reported, for the first time, the calcite precipitation by P. psycrodurans and R. qingshengii. Optical microscopy and Scanning Electron Microscopy showed crystals with irregular and spherical shapes, and beige and white colours. Furthermore, crystals formation appeared to be strain-specific. X-Ray diffraction analysis confirmed crystals composition as CaCO3. Biocementation tests showed that MICP treatments of mortar cubes using P. psycrodurans caused an increase in their compressive strength compared to control samples. The positive action of a native MICP strain in mortar blocks biomineralization is shown, which is of great interest and potential for the construction industry.

Gas-diffusion microextraction coupled with spectrophotometry for the determination of formaldehyde in cork agglomerates.

In this work, a simple methodology was developed for the extraction and determination of free formaldehyde content in cork agglomerate samples. For the first time, gas-diffusion microextraction was used for the extraction of volatile formaldehyde directly from samples, with simultaneous derivatization with acetylacetone (Hantzsch reaction). The absorbance of the coloured solution was read in a spectrophotometer at 412 nm. Different extraction parameters were studied and optimized (extraction temperature, sample mass, volume of acceptor solution, extraction time and concentration of derivatization reagent) by means of an asymmetric screening. The developed methodology proved to be a reliable tool for the determination of formaldehyde in cork agglomerates with the following suitable method features: low LOD (0.14 mg kg-1) and LOQ (0.47 mg kg-1), r 2 = 0.9994, and intraday and interday precision of 3.5 and 4.9%, respectively. The developed methodology was applied to the determination of formaldehyde in different cork agglomerate samples, and contents between 1.9 and 9.4 mg kg-1 were found. Furthermore, formaldehyde was also determined by the standard method EN 717-3 for comparison purposes; no significant differences between the results of both methods were observed. Graphical abstract Representation of the GDME system and its main components.

Persistence of pentolite (PETN and TNT) in soil microcosms and microbial enrichment cultures.

Pentolite is a mixture (1:1) of 2,4,6-trinitrotoluene (TNT) and pentaerythritol tetranitrate (PETN), and little is known about its fate in the environment. This study was aimed to determine the dissipation of pentolite in soils under laboratory conditions. Microcosm experiments conducted with two soils demonstrated that dissipation rate of PETN was significantly slower than that of TNT. Interestingly, the dissipation of PETN was enhanced by the presence of TNT, while PETN did not enhanced the dissipation of TNT. Pentolite dissipation rate was significantly faster under biostimulation treatment (addition of carbon source) in soil from the artificial wetland, while no such stimulation was observed in soil from detonation field. In addition, the dissipation rate of TNT and PETN in soil from artificial wetland under biostimulation was significantly faster than the equivalent abiotic control, although it seems that non-biological processes might also be important for the dissipation of TNT and PETN. Transformation of PETN was also slower during establishment of enrichment culture using pentolite as the sole nitrogen source. In addition, transformation of these explosives was gradually reduced and practically stopped after the forth cultures transfer (80 days). DGGE analysis of bacterial communities from these cultures indicates that all consortia were dominated by bacteria from the order Burkholderiales and Rhodanobacter. In conclusion, our results suggest that PETN might be more persistent than TNT.

Environmental occurrence of arsenic in Colombia: a review.

The international literature on the presence of arsenic (As) in Latin America does not disclose the true magnitude of the presence of As in Colombia. In this paper, we summarize the literature on As occurrence in Colombia. The data reveal that As is present in matrices such as soil, sediments and water and in the food chain. Some of the As concentrations exceed the limits specified by national and international regulations. Arsenic higher concentrations are associated with mining regions (e.g., soils, up to 148 mg/kg; sediments, up to 1400 mg/kg) and agricultural areas (e.g., vegetables, up to 5.40 mg/kg; irrigation water, up to 255 µg/L), and underscore the potential human and environmental risks associated with the presence of As in the country. This review highlights the importance of focusing research on understanding the occurrence, origin and distribution of As in Colombia to better understand its environmental and public health impact.

Di-µ(2)-chlorido-dichloridoocta-methyldi-µ(3)-oxido-tetra-tin(IV) bis[chloridodimeth-yl(pyrrolidine-1-carbodithio-ato-κ(2)S,S')tin(IV)].

In the title co-crystal, [Sn(4)(CH(3))(8)Cl(4)O(2)]·2[Sn(CH(3))(2)Cl(C(4)H(8)NS(2))], all the Sn(IV) atoms are in distorted trigonal-bipyramidal environments. In the mononuclear species, the carbodithio-ate ligand is unsymmetrically coordinated to the Sn(IV) atom, with Sn-S distances of 2.6722 (12) and 2.4706 (11) Å. All atoms with the exception of the methyl groups and one of the pyrrolidine ring CH(2) groups lie on a crystallographic mirror plane. The pyrrolidine ring exhibits an envelope conformation; the C atom at the flap is disordered above and below the plane of symmetry with fixed occupation factors of 0.50. The centrosymmetric dimer species consists of a central Sn(2)O(2) unit with two adjacent Sn(2)OCl four-membered rings.

Nitrile hydrolysing activities of deep-sea and terrestrial mycolate actinomycetes.

Nitrile metabolising actinomycetes previously recovered from deep-sea sediments and terrestrial soils were investigated for their nitrile transforming properties. Metabolic profiling and activity assays confirmed that all strains catalysed the hydrolysis of nitriles by a nitrile hydratase/amidase system. Acetonitrile and benzonitrile, when used as growth substrates for enzyme induction experiments, had a significant influence on the biotransformation activities towards various nitriles and amides. The specific activities of selected deep-sea and terrestrial acetonitrile-grown bacteria against a suite of nitriles and amides were higher than those of the only other reported marine nitrile-hydrolysing R. erythropolis, isolated from a shallow sediment. The increase of nitrile chain length appeared to have negative influence on the nitrile hydratase activity of acetonitrile-grown bacteria, but the same was not true for benzonitrile-grown bacteria. The nitrile hydratases and amidases were constitutive in 10 of the 16 deep-sea and terrestrial actinomycetes studied, and one strain showed an inducible hydratase and a constitutive amidase. Most of the deep-sea strains had constitutive activities and showed some of the highest activities and broadest substrate specificities of organisms included in this study.

Diversity of nitrile hydratase and amidase enzyme genes in Rhodococcus erythropolis recovered from geographically distinct habitats.

A molecular screening approach was developed in order to amplify the genomic region that codes for the alpha- and beta-subunits of the nitrile hydratase (NHase) enzyme in rhodococci. Specific PCR primers were designed for the NHase genes from a collection of nitrile-degrading actinomycetes, but amplification was successful only with strains identified as Rhodococcus erythropolis. A hydratase PCR product was also obtained from R. erythropolis DSM 43066(T), which did not grow on nitriles. Southern hybridization of other members of the nitrile-degrading bacterial collection resulted in no positive signals other than those for the R. erythropolis strains used as positive controls. PCR-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the hydratases in the R. erythropolis strains revealed unique patterns that mostly correlated with distinct geographical sites of origin. Representative NHases were sequenced, and they exhibited more than 92.4% similarity to previously described NHases. The phylogenetic analysis and deduced amino acid sequences suggested that the novel R. erythropolis enzymes belonged to the iron-type NHase family. Some different residues in the translated sequences were located near the residues involved in the stabilization of the NHase active site, suggesting that the substitutions could be responsible for the different enzyme activities and substrate specificities observed previously in this group of actinomycetes. A similar molecular screening analysis of the amidase gene was performed, and a correlation between the PRS patterns and the geographical origins identical to the correlation found for the NHase gene was obtained, suggesting that there was coevolution of the two enzymes in R. erythropolis. Our findings indicate that the NHase and amidase genes present in geographically distinct R. erythropolis strains are not globally mixed.

Survivability of bacteria ejected from icy surfaces after hypervelocity impact.

Both the Saturnian and Jovian systems contain satellites with icy surfaces. If life exists on any of these icy bodies (in putative subsurface oceans for example) then the possibility exists for transfer of life from icy body to icy body. This is an application of the idea of Panspermia, wherein life migrates naturally through space. A possible mechanism would be that life, here taken as bacteria, could become frozen in the icy surface of one body. If a high-speed impact occurred on that surface, ejecta containing the bacteria could be thrown into space. It could then migrate around the local region of space until it arrived at a second icy body in another high-speed impact. In this paper we consider some of the necessary steps for such a process to occur, concentrating on the ejection of ice bearing bacteria in the initial impact, and on what happens when bacteria laden projectiles hit an icy surface. Laboratory experiments using high-speed impacts with a light gas gun show that obtaining icy ejecta with viable bacterial loads is straightforward. In addition to demonstrating the viability of the bacteria carried on the ejecta, we have also measured the angular and size distribution of the ejecta produced in hypervelocity impacts on ice. We have however been unsuccessful at transferring viable bacteria to icy surfaces from bacteria laden projectiles impacting at hypervelocities.

Discrimination and taxonomy of geographically diverse strains of nitrile-metabolizing actinomycetes using chemometric and molecular sequencing techniques.

Mycolic acid-containing actinomycetes capable of metabolizing nitriles were recovered from deep-sea sediments and terrestrial soils by enrichment culture on acetonitrile, benzonitrile, succinonitrile or bromoxynil. A total of 43 nitrile-degrading strains were isolated and, together with previously recovered nitrile-degrading rhodococci, were identified by a polyphasic taxonomic approach, which included mycolic acid profiles, pyrolysis mass spectrometry (PyMS), genomic fingerprinting based on sequence variability of the 16S ribosomal RNA gene using polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism, and 16S rRNA gene sequence comparison. Isolates phylogenetically related to Rhodococcus erythropolis dominated the culturable microorganisms from most marine and terrestrial samples. These isolates clustered together in a major pyrogroup that showed high congruence with PRS profiles of the 16S rRNA gene. Such high congruence also was obtained for other recovered isolates that were assigned to species of Rhodococcus and Gordonia. Sequencing data validated the results obtained by PRS analysis and enabled phylogenetic relationships to be established. Some of the recovered bacteria probably represent novel microbial species. The fact that nitrile-metabolizing microorganisms were recovered from a wide range of habitat types suggests that nitrile transforming enzymatic activity is geographically widely distributed in nature.