Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates

Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.

Gene expression analysis of Alcaligenes faecalis during induction of heterotrophic nitrification

Alcaligenes faecalis is a heterotrophic nitrifying bacterium that oxidizes ammonia and generates nitrite and nitrate. When A. faecalis was cultivated in a medium containing pyruvate and ammonia as the sole carbon and nitrogen sources, respectively, high concentrations of nitrite accumulated in the medium whose carbon/nitrogen (C/N) ratio was lower than 10 during the exponential growth phase, while the accumulation was not observed in the medium whose C/N ratio was higher than 15. Comparative transcriptome analysis was performed using nitrifying and non-nitrifying cells of A. faecalis cultivated in media whose C/N ratios were 5 and 20, respectively, to evaluate the fluctuations of gene expression during induction of heterotrophic nitrification. Expression levels of genes involved in primary metabolism did not change significantly in the cells at the exponential growth phase under both conditions. We observed a significant increase in the expression levels of four gene clusters: pod cluster containing the gene encoding pyruvic oxime dioxygenase (POD), podh cluster containing the gene encoding a POD homolog (PODh), suf cluster involved in an iron-sulfur cluster biogenesis, and dnf cluster involved in a novel hydroxylamine oxidation pathway in the nitrifying cells. Our results provide valuable insight into the biochemical mechanism of heterotrophic nitrification.

A Metagenomic Nanopore Sequence Analysis Combined with Conventional Screening and Spectroscopic Methods for Deciphering the Antimicrobial Metabolites Produced by Alcaligenes faecalis Soil Isolate MZ921504

The continuous development of multidrug resistance pathogens with limited therapeutic options has become a great problem globally that impose sever health hazards. Accordingly, searching for of new antimicrobials became an urgent demand and great challenge. Soil significantly have been associated with several species that are antibiotic producers. In this study, combination of conventional screening methods with Liquid chromatography- Mass spectroscopy (LC/MS) and metagenomic nanopore sequence analysis have been conducted for the deciphering the active metabolites produced by soil isolate(s). Preliminary soil screening resulted in a Gram-negative isolate identified via 16S ribosomal RNA as Alcaligenes faecalis isolate MZ921504 with promising antimicrobial activities against wide range of MDR gram-positive and gram-negative pathogens. The LC/MS analysis of the metabolites of A. faecalis isolate MZ921504 confirmed the presence of ectoine, bacillibactin, quinolobactin and burkholderic acid. Metagenomics sequence analysis of the soil sample (NCBI GenBank accession PRJNA771993) revealed the presence of conserved biosynthetic gene clusters of ectoine, bacteriocin, bacillibactin, quinolobactin, terpene and burkholderic acid of A. faecalis. In conclusion, A. faecalis isolate MZ921504 is a promising source for antimicrobial metabolites. LC/MS spectral analysis and third generation sequencing tools followed by secondary metabolite gene clusters analysis are useful methods to predict the nature of the antimicrobial metabolites.

Optimization of hemolysin formation in Alcaligenes species isolated from abattoir wastewater samples in Akure, Ondo State, Nigeria

Hemolysin is significantly toxic and it is used as a molecular marker for pathogenicity. This study evaluates the conditions for optimal hemolysin production by Alcaligenes faecalis strains isolated from a city abattoir wastewater. The parameters investigated for hemolysin formation were size of the inoculum, initial pH of production medium, bacterial incubation temperature, agitation speed and growth media. Thereafter, the effect by various parameter on the hemolytic activity for the formation of hemolysin were assessed. The genus Alcaligenes was assigned to the test organisms after analyzing their 16S rRNA gene sequence with accession numbers: MF498824, MF498825 and MF498827. Optimum conditions for hemolysin formation in Alcaligenes faecalis strain OS42 were inoculum size of 0.5% (v/v), pH 9, 20 oC, 0 rpm and brain heart infusion broth. Hemolytic activities, 77% and 79% were achieved at 20 h for strains OS42 and OS61, respectively. Cholesterol and ethylenediaminetetraacetic acid did not affect hemolysin formation. This work revealed that the hemolysin formation in Alcaligenes strains was sourced from abattoir wastewater effluent and the effluent was contaminated with pathogenic Alcaligenes strains which is a public health hazard due to their prospective infection to human and animal.

Complete Genome Analysis of a Novel Alcaligenes Faecalis Phage vB_Af_QDWS595

A novel lytic phage named vB_Af_QDWS595 against Alcaligenes faecalis was isolated and characterized in this study. The genome of phage vB_Af_QDWS595 was sequenced and analyzed, and the result revealed that the phage contained a 88,795 bp of circular double-stranded DNA with 41.12% of GC content. There were 74 putative open reading frames (ORFs) and 11 tRNAs predicted in genome of phage vB_Af_QDWS595. Phenotype and phylogeny analysis indicated that this phage might be a new member within the family Schitoviridae. Phage vB_Af_QDWS595 is the first sequenced phage against Alcaligenes faecalis to the best of our knowledge.

Physicochemical and molecular characterization of heavy metal–tolerant bacteria isolated from soil of mining sites in Nigeria

Background Mining for precious metals is detrimental to the composition of soil structure and microbial diversity distribution and is a health risk to human communities around the affected communities. This study was aimed at determining the physical and chemical characteristics and diversity of bacteria in the soil of local mining sites for biosorption of heavy metals. Results Results of physical and chemical characteristics showed mean pH values and percentage organic carbon to range from 7.1 to 8.2 and 0.18 to 1.12% respectively with statistical significance between sampling sites (P ≤ 0.05). Similarly, cation exchange capacity, electrical conductivity, moisture, total nitrogen, and carbon/nitrogen ratio (C:N) in the soil ranged between 1.52 to 3.57 cmol/kg, 0.15 to 0.32 ds/m, 0.14 to 0.82%, 0.10 to 0.28%, and 1.7 to 4.8 respectively. The highest heavy metal concentration of 59.01 ppm was recorded in soils obtained from site 3. The enumeration of viable aerobic bacteria recorded the highest mean count of 4.5 × 106 cfu/g observed at site 2 with statistical significance (P ≤ 0.05) between the sampled soils. Alcaligenes faecalis strain UBI, Aeromonas sp. strain UBI, Aeromonas sobria, and Leptothrix ginsengisoli that make up 11.2% of total identified bacteria were able to grow in higher amended concentrations of heavy metals. The evolutionary relationship showed the four heavy metal–tolerant bacteria identified belonged to the phylum Proteobacteria of class Betaproteobacteria in the order Burkholderiales. Heavy metal biosorption by the bacteria showed Alcaligenes faecalis strain UBI having the highest uptake capacity of 73.5% for Cu. Conclusion In conclusion, Alcaligenes faecalis strain UBI (MT107249) and Aeromonas sp. strain UBI (MT126242) identified in this study showed promising capability to withstand heavy metals and are good candidates in genetic modification for bioremediation.

Whole-Genome Sequencing of Alcaligenes faecalis HZ01, with Potential to Inhibit Nontuberculous Mycobacterial Growth

Alcaligenes faecalis is a Gram-negative rod that is ubiquitous in the environment and is an opportunistic human pathogen. Here, we report the whole-genome sequencing analysis of A. faecalis HZ01, which presents mycobacterial growth inhibitory activity and was isolated from a contaminated culture of Mycobacterium chubuense ATCC 27278.