Exploring new marine bacterial species, Alcaligenes faecalis Alca F2018 valued for bioconversion of shrimp chitin to chitosan for concomitant biotechnological applications.

The exploitation of chitinous materials seems to be an infinite treasure. To this end, using shellfish waste as the sole carbon/nitrogen source solves environmental challenges while lowering microbial chitinase production costs. Bioconversion of shellfish chitin wastes such as shrimp shells has recently been investigated for the production of enzymes and bioactive materials in order to maximize the utilization of chitin-containing seafood processing wastes. In this study, the bioconversion of chitin to chitosan by Alcaligenes faecalis Alca F2018 revealed the highest chitin deacetylase (CDA) activity of 40.6 U/µg. The resulted low Km and high Vmax values explain the high affinity of the purified CDA to the p-nitroacetanilide substrate. CDA with a molecular weight of 66 KDa was purified from F2018 strain, with a 14.5% yield. FT-IR revealed distinct chitosan peaks and XRD revealed that chitosan samples had lower crystallinity than chitin. TGA analysis revealed that the recovered chitosan samples were more thermally stable. The deacetylation degree percentages of the produced chitosan are in the same range as that of the commercial chitosan, suggesting the promising potential of A. faecalis Alca F2018 to utilize shrimp shells in their raw form in the fermentation media based on its CDA enzyme activity.

Modulation in phenolic root exudate profile of Abelmoschus esculentus expressing activation of defense pathway.

Phenolics play a key role in communication between plants and microbes in the rhizosphere. In this study, shikimic, gallic, fumaric, ferulic, vanillic acid and quercetin in root exudates of Abelmoschus esculentus act as chemoattractants of endophytic Alcaligenes faecalis strains, BHU 12, BHU 16 and BHU M7. In vitro chemotaxis assay showed that BHU 12 expressed highest chemotactic movement (CFU ∼50×1012) towards A.esculentus root exudates followed by BHU 16 and BHU M7 (CFU∼ 9×1012), thereby confirming their ability to colonize the host rhizoplane region. However, BHU 16 expressed highest biofilm formation ability followed by BHU 12 and BHU M7. Assessment of chemotactic and biofilm formation potential towards individual phenolic acids revealed BHU 12 to be maximally attracted towards 1µM shikimic acid (2×1015) while BHU 16 towards 1mM vanillic acid (6.5×1012) and BHU M7 towards 1mM ferulic acid (3.5×1012), thereby confirming the phenolic acid components responsible for particularly attracting the endophytic isolates. Upon colonization, the endophytic isolates modified the phenolic profiles of root exudates in planta in a manner so as to plausibly attract more of the beneficial rhizospheric microbiota as well as self-fortification against pathogenic microbes. This hypothesis was verified by monitoring the changes in phenolic components of A. esculentus root exudate owing to S. rolfsii infection, a disastrous soil-borne pathogen. Thus, on the whole, the work provides intricate details of plant-endophyte interactions for biotic stress management through careful manipulation of root exudates, thereby aiding in sustainable agriculture.

Biodegradation of ochratoxin A by Alcaligenes faecalis isolated from soil.

AIMS: The aim of this study is to report on the hydrolytic action of Alcaligenes faecalis isolated from soil samples and its ability to degrade ochratoxin A. METHODS AND RESULTS: An A. faecalis strain was identified and characterized by employing both a phenotypic analysis and 16S rDNA sequence analysis. The results show that this strain could degrade ochratoxin A efficiently but could not use it as a sole carbon source. Ochratoxin α was confirmed as a degradation product in the intracellular extract of A. faecalis using UPLC-MS/MS. Our results suggest that the biodegradation of ochratoxin A by the A. faecalis strain occurs through the hydrolysis of the ochratoxin A amide bond by a putative peptidase. This is the first report to date on the degradation of ochratoxin A by A. faecalis. CONCLUSION: The A. faecalis strain is presumably a suitable candidate for use in the biodegradation of ochratoxin A. SIGNIFICANCE AND IMPACT OF THE STUDY: Ochratoxin A, which is produced by some filamentous fungi, severely impacts human and animal health by contaminating several types of food and feed. Our study contributes to the identification of the function of A. faecalis 0D-1, which is capable of producing hydrolytic enzyme(s) to biodegrade ochratoxin A into nontoxic ochratoxin α, to minimize the risk associated with ochratoxin A exposure.

Alcaligenes faecalis ZD02, a Novel Nematicidal Bacterium with an Extracellular Serine Protease Virulence Factor.

Root knot nematodes (RKNs) are the world's most damaging plant-parasitic nematodes (PPNs), and they can infect almost all crops. At present, harmful chemical nematicides are applied to control RKNs. Using microbial nematicides has been proposed as a better management strategy than chemical control. In this study, we describe a novel nematicidal bacterium named Alcaligenes faecalis ZD02. A. faecalis ZD02 was isolated from Caenorhabditis elegans cadavers and has nematostatic and nematicidal activity, as confirmed by C. elegans growth assay and life span assay. In addition, A. faecalis ZD02 fermentation broth showed toxicity against C. elegans and Meloidogyne incognita. To identify the nematicidal virulence factor, the genome of strain ZD02 was sequenced. By comparing all of the predicted proteins of strain ZD02 to reported nematicidal virulence factors, we determined that an extracellular serine protease (Esp) has potential to be a nematicidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita. Using C. elegans as the target model, we found that both A. faecalis ZD02 and the virulence factor Esp can damage the intestines of C. elegans. The discovery that A. faecalis ZD02 has nematicidal activity provides a novel bacterial resource for the control of RKNs.

Tributyltin chloride (TBTCl)-enhanced exopolysaccharide and siderophore production in an estuarine Alcaligenes faecalis strain.

Tributyltin chloride (TBTCl) has been used extensively as an antifouling agent in ship paints, which results in the contamination of aquatic sites. These contaminated sites serve as enrichment areas for TBTCl-resistant bacterial strains. One TBTCl-resistant bacterial strain was isolated from the sediments of Zuari estuary, Goa, India, which is a major hub of various ship-building activities. Based on biochemical characteristics and 16S rDNA sequence analysis, this bacterial strain was identified as Alcaligenes faecalis and designated as strain SD5. It could degrade ≥3 mM TBTCl by using it as a sole carbon source and transform it into the less toxic dibutyltin chloride, which was confirmed by nuclear magnetic resonance and mass spectroscopy. Interestingly, this bacterial strain also showed enhanced exopolysaccharide and siderophore production when cells were exposed to toxic levels of TBTCl, suggesting their involvement in conferring resistance to this antifouling biocide as well as degradative capability respectively.

Characterization of Alcaligenes faecalis strain AD15 indicating biocontrol activity against plant pathogens.

Bacterial strain possessing both bacteriostatic and fungistatic activity (biocontrol activity) against pathogens of cyclamen (Cyclamen sp.) was isolated from the soil in Gifu Prefecture, Japan, and characterized with respect to its taxonomic and biocontrol properties. The sequence of its 16S rRNA gene, morphology, biochemistry, and fatty acid composition demonstrated that it is a strain most closely related to Alcaligenes faecalis subsp. faecalis LMG 1229(T). The isolate was named A. faecalis strain AD15. A. faecalis AD15 produced hydroxylamine at maximum yields of 33.3±1.7 mg/L after 16 h cultivation in LB medium and 19.0±0.44 mg/L after 19 h cultivation in synthetic medium. Moreover, minimum inhibitory concentrations of hydroxylamine against the cyclamen pathogens Pantoea agglomerans and Colletotrichum gloeosporioides were 4.20±0.98 and 16.5±0.67 mg/L. These results indicated that the biocontrol activity of strain AD15 might be attributed to hydroxylamine, a metabolite in the culture medium, and it had the potential for biopesticide application.

Isolation and characterization of a heterotrophic nitrifier from coke plant wastewater.

This study investigated some factors affecting ammonium removal and nitrite accumulation by Alcaligenes faecalis C16, which was isolated from the activated sludge of a coking wastewater treatment plant. Nitrite was produced from ammonium only in the presence of citrate, acetate, meat extract, peptone or ethanol. The highest amount of nitrite was found with citrate as carbon source. A. faecalis C16 could not use glucose, fructose, sucrose and methanol. Under the optimum conditions of initial pH 6.0, C/N 14, 30 °C and 120 rpm, a maximum nitrite accumulation of 28.29 mg/L NO(2)(-)-N was achieved when the organism grew with citrate in four days. Nitrite accumulation increased with the increase of NH(4)(+)-N. Furthermore, A. faecalis C16 was shown to have phenol-degrading capacity during ammonium removal. Metabolism of phenol resulted in acidification of the media, which is not favorable for nitrification, whereas many other carbon sources made the medium more alkaline. However, no inhibitory effect by phenol was observed when phenol and acetate were used as mixed carbon source at different phenol/sodium acetate (P/S) ratios and their pH values were all controlled above 9.2 or P/S ratios below 5:5. These results suggested that A. faecalis C16 has some potential application in industrial wastewater treatment systems.

Enantioselective biocatalytic hydrolysis of (R,S)-mandelonitrile for production of (R)-(-)-mandelic acid by a newly isolated mutant strain.

(R)-(-)-Mandelic acid (R-MA) is an important intermediate with broad uses. Recently, R-MA production using nitrilase has been gaining more and more attention due to its higher productivity and enantioselectivity. In this work, a new bacterium WT10, which exhibited favorable nitrilase activity and excellent enantioselectivity for production of R-MA by enantioselective biocatalytic hydrolysis of (R,S)-mandelonitrile, was isolated and identified as a strain of Alcaligenes faecalis. In order to improve its nitrilase activity for industrial application, the wild-type strain WT10 was further subjected to mutagenesis using a combined LiCl-ultraviolet irradiation and low energy N(+) ion beams implantation technique. A valuable mutant strain A. faecalis ZJUTB10 was obtained. The nitrilase specific activity of the mutant strain was greatly improved up to 350.8 U g(-1), in comparison with wild-type strain WT10 of 53.09 U g(-1). The reaction conditions for R-MA production by mutant strain A. faecalis ZJUTB10 were also optimized. Nitrilase activity in mutant strain showed a broad pH optimum at pH 7.7-8.5. The optimal temperature was 35°C. The highest production rate reached 9.3 mmol h(-1) g(-1). The results showed that mutant strain A. faecalis ZJUTB10 was a new candidate for efficient R-MA production from (R,S)-mandelonitrile and could potentially be used in industrial production.

Potential pathogenic bacteria in metalworking fluids and aerosols from a machining facility.

The metalworking and machining industry utilizes recirculating metalworking fluids for integral aspects of the fabrication process. Despite the use of biocides, these fluids sustain substantial biological growth. Subsequently, the high-shear forces incurred during metalworking processing aerosolize bacterial cells and may cause dermatologic and respiratory effects in exposed workers. We quantified and identified the bacterial load for metalworking fluid and aerosol samples of a machining facility in the US Midwest during two seasons. To investigate the presence of potentially pathogenic bacteria in fluid and air, we performed 16S rRNA gene surveys. The concentration of total bacterial cells (including culturable and nonculturable cells) was relatively constant throughout the study, averaging 5.1 × 108 cells mL⁻¹ in the fluids and 4.8 × 105 cells m⁻³ in the aerosols. We observed bacteria of potential epidemiologic significance from several different bacterial phyla in both fluids and aerosols. Most notably, Alcaligenes faecalis was identified through both direct sequencing and culturing in every sample collected. Elucidating the bacterial community with gene surveys showed that metalworking fluids were the source of the aerosolized bacteria in this facility.

Improvement in ammonium removal efficiency in wastewater treatment by mixed culture of Alcaligenes faecalis no. 4 and L1.

To improve ammonium removal efficiency in wastewater treatment, a mixed culture of Alcaligenes faecalis no. 4 and its mutant L1, both of which have heterotrophic nitrification and aerobic denitrification abilities, was performed. In a batch culture, no. 4 has a higher denitrification ability than L1, but its ammonium removal rate was lower. In a mixed continuous culture in the ammonium loading range of 750 to 3500 mg-N/l/d, the average ammonium removal rate and the average denitrification ratio were 61 mg-N/l/h and 31%, respectively. In the mixed culture, the ammonium removal rate was twofold higher than that in a single culture of no. 4, the rate was similar to that in a single culture of L1, and the denitrification ratio was very high compared with that in the single culture of L1.

Fluorescence in situ hybridization for rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis recovered from cystic fibrosis patients.

Achromobacter xylosoxidans is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients, but identification with biochemical tests is unreliable. We describe fluorescence in situ hybridization assays for the rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis. Both assays showed high sensitivities and high specificities with a collection of 155 nonfermenters from CF patients.

The sulfonated osmolyte N-methyltaurine is dissimilated by Alcaligenes faecalis and by Paracoccus versutus with release of methylamine.

Selective enrichments yielded bacterial cultures able to utilize the osmolyte N-methyltaurine as sole source of carbon and energy or as sole source of fixed nitrogen for aerobic growth. Strain MT1, which degraded N-methyltaurine as a sole source of carbon concomitantly with growth, was identified as a strain of Alcaligenes faecalis. Stoichiometric amounts of methylamine, whose identity was confirmed by matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry, and of sulfate were released during growth. Inducible N-methyltaurine dehydrogenase, sulfoacetaldehyde acetyltransferase (Xsc) and a sulfite dehydrogenase could be detected. Taurine dehydrogenase was also present and it was hypothesized that taurine dehydrogenase has a substrate range that includes N-methyltaurine. Partial sequences of a tauY-like gene (encoding the putative large component of taurine dehydrogenase) and an xsc gene were obtained by PCR with degenerate primers. Strain N-MT utilized N-methyltaurine as a sole source of fixed nitrogen for growth and could also utilize the compound as sole source of carbon. This bacterium was identified as a strain of Paracoccus versutus. This organism also expressed inducible (N-methyl)taurine dehydrogenase, Xsc and a sulfite dehydrogenase. The presence of a gene cluster with high identity to a larger cluster from Paracoccus pantotrophus NKNCYSA, which is now known to dissimilate N-methyltaurine via Xsc, allowed most of the overall pathway, including transport and excretion, to be defined. N-Methyltaurine is thus another compound whose catabolism is channelled directly through sulfoacetaldehyde.

Isolation and characterization of a chlorpyrifos and 3,5,6-trichloro-2-pyridinol degrading bacterium.

A bacterium, isolated from contaminated soils around a chemical factory and named strain DSP3 was capable of biodegrading both chlorpyrifos and 3,5,6-trichloro-2-pyridinol. Based on the results of phenotypic features, phylogenetic similarity of 16S rRNA gene sequences, DNA G+C content, and DNA homology between strain DSP3 and reference strains, strain DSP3 was identified as Alcaligenes faecalis. Chlorpyrifos was utilized as the sole source of carbon and phosphorus by strain DSP3. We examined the role of strain DSP3 in the degradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol under different culture conditions. Parathion and diazinon could also be degraded by strain DSP3 when provided as the sole sources of carbon and phosphorus. An addition of strain DSP3 (10(8)cells g(-1)) to soil with chlorpyrifos (100 mg kg(-1)) resulted in a higher degradation rate than the one obtained from non-inoculated soils. Different degradation rates of chlorpyrifos in six types of treated soils suggested that soils used for cabbage growing in combination with inoculation of strain DSP3 showed enhanced microbial degradation of chlorpyrifos.

Alcaligenes faecalis subsp. phenolicus subsp. nov. a phenol-degrading, denitrifying bacterium isolated from a graywater bioprocessor.

A Gram (-) coccobacillary bacterium, J(T), was isolated from a graywater bioprocessor. 16S rRNA and biochemical analysis has revealed strain J(T) closely resembles Alcaligenes faecalis ATCC 8750T and A. faecalis subsp. parafaecalis DSM 13975T, but is a distinct, previously uncharacterized isolate. Strain J(T), along with the type strain of A. faecalis and its previously described subspecies share the ability to aerobically degrade phenol. The degradation rates of phenol for strain J(T) and reference phenol degrading bacteria were determined by photometrically measuring the change in optical density when grown on 0.1% phenol as the sole carbon source, followed by addition of Gibb's reagent to measure depletion of substrate. The phenol degradation rates of strain J(T) was found to exceed that of the phenol hydroxylase group III bacterium Pseudomonas pseudoalcaligenes, with isolate J(T) exhibiting a doubling time of 4.5 h. The presence of the large subunit of the multicomponent phenol hydroxylase gene in strain J(T) was confirmed by PCR. The presence of the nirK nitrite reductase gene as demonstrated by PCR as well as results obtained from nitrite media indicated denitrification at least to N2O. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA DNA hybridization, we propose assigning a novel subspecies of Alcaligenes faecalis, to be named Alcaligenes faecalis subsp. phenolicus with the type strain J(T) (= DSM 16503) (= NRRL B-41076).

Kerstersia gyiorum gen. nov., sp. nov., a novel Alcaligenes faecalis-like organism isolated from human clinical samples, and reclassification of Alcaligenes denitrificans Rüger and Tan 1983 as Achromobacter denitrificans comb. nov.

A polyphasic taxonomic study was performed on nine isolates recovered from various human clinical samples. Phenotypically, these isolates resembled Alcaligenes faecalis. Whole-cell protein analysis distinguished two different species, and this was confirmed by DNA-DNA hybridizations. Cellular fatty acid analysis and 16S rDNA sequence analysis indicated that these isolates were related to the genera Alcaligenes, Bordetella, Achromobacter and Pigmentiphaga and belonged to the family Alcaligenaceae. On the basis of the results of this study, the organisms were classified in a novel genus, Kerstersia gen. nov. This genus comprises one species, Kerstersia gyiorum sp. nov. (type strain LMG 5906(T)=API 184-2-84(T)=CCUG 47000(T)), and several unnamed isolates. The DNA G+C content of members of the genus Kerstersia is between 61.5 and 62.9 mol%. On the basis of previously published DNA-DNA hybridization results and data from chemotaxonomic studies, it is proposed that Alcaligenes denitrificans Rüger and Tan 1983 be reclassified as Achromobacter denitrificans comb. nov.