Re-considering and designing microbiomes for future waste biorefinery.

It is an increasingly promising research direction using microbiomes to produce various chemicals in order to support people's growing need for sustainability. Currently, bottom-up constructed defined microbiomes and top-down constructed undefined microbiomes play an essential role in the fields of synthetic biology and environmental engineering, respectively. However, if we are goal-oriented and want to align scientific principles with technology and engineering in future waste biorefinery, we need to reconsider and design microbiomes interdisciplinarily. In this editorial, we briefly review the latest applications of two approaches to microbiome design (bottom-up and top-down) and the dilemmas faced in using complex waste. Consequently, we introduce the concept of 'sustainable synthetic microbiomics' to apply combined bottom-up and top-down constructed microbiomes to provide products for human needs from low-value waste. Furthermore, we outline the relatively comprehensive research contents and expected prospects based on the pressing problems. Finally, burning questions on key research contents are proposed for specific cases, hoping to provide valuable views for future microbiome biorefinery.

A discussion and evaluation of statistical procedures used by JIMB authors when comparing means.

Out of the 166 articles published in Journal of Industrial Microbiology and Biotechnology (JIMB) in 2019-2020 (not including special issues or review articles), 51 of them used a statistical test to compare two or more means. The most popular test was the (Standard) t-test, which often was used to compare several pairs of means. Other statistical procedures used included Fisher's least significant difference (LSD), Tukey's honest significant difference (HSD), and Welch's t-test; and to a lesser extent Bonferroni, Duncan's Multiple Range, Student-Newman-Keuls, and Kruskal-Wallis tests. This manuscript examines the performance of some of these tests with simulated experimental data, typical of those reported by JIMB authors. The results show that many of the most common procedures used by JIMB authors result in statistical conclusions that are prone to have large false positive (Type I) errors. These error-prone procedures included the multiple t-test, multiple Welch's t-test, and Fisher's LSD. These multiple comparisons procedures were compared with alternatives (Fisher-Hayter, Tukey's HSD, Bonferroni, and Dunnett's t-test) that were able to better control Type I errors. NON-TECHNICAL SUMMARY: The aim of this work was to review and recommend statistical procedures for Journal of Industrial Microbiology and Biotechnology authors who often compare the effect of several treatments on microorganisms and their functions.

Effective application of immobilized second generation industrial Saccharomyces cerevisiae strain on consolidated bioprocessing.

Integrated bioprocessing strategies can facilitate ethanol production from both cellulose and hemicellulose fractions of lignocellulosic biomass. Consolidated bioprocessing (CBP) is an approach that combines enzyme production, biomass hydrolysis and sugar fermentation in a single step. However, technologies that propose the use of microorganisms together with solid biomass present the difficulty of the recovery and reuse of the biocatalyst, which can be overcome by cell immobilization. In this regard, this work applied immobilized cells of AC14 yeast, a recombinant yeast that secretes 7 hydrolytic enzymes, in the CBP process in a successful proof-of-concept for the enzyme access to the substrate polymers. The most appropriate cell load for CBP under the conditions studied with immobilized cells was selected among three optical densities (OD) 10, 55 and 100. These experiments were performed with free cells to ensure that the results were not biased by mass limitations effects. OD 10 achieved 100% of the sugar consumption and the higher specific production of enzymes, being selected for further studies. Diffusional effects were observed with immobilized cells under static conditions. However, mass transfer limitations were mitigated under agitation, with an 18.5% increase in substrate consumption rate (from 2.7 to 3.5 g/L/h), reaching the same substrate uptake rates as free cells. In addition, immobilized cells achieved 100% hydrolysis and consumption of all substrates offered within only 12 h. Overall, this is the first report of a successful application of immobilized yeast cells in CBP processes for bioethanol production, a promising technology that can be extended to other biorefinery bioproducts.

Dung beetle-associated yeasts display multiple stress tolerance: a desirable trait of potential industrial strains.

BACKGROUND: Stress-tolerant yeasts are highly desirable for cost-effective bioprocessing. Several strategies have been documented to develop robust yeasts, such as genetic and metabolic engineering, artificial selection, and natural selection strategies, among others. However, the significant drawbacks of such techniques have motivated the exploration of naturally occurring stress-tolerant yeasts. We previously explored the biodiversity of non-conventional dung beetle-associated yeasts from extremophilic and pristine environments in Botswana (Nwaefuna AE et.al., Yeast, 2023). Here, we assessed their tolerance to industrially relevant stressors individually, such as elevated concentrations of osmolytes, organic acids, ethanol, and oxidizing agents, as well as elevated temperatures. RESULTS: Our findings suggest that these dung beetle-associated yeasts tolerate various stresses comparable to those of the robust bioethanol yeast strain, Saccharomyces cerevisiae (Ethanol Red™). Fifty-six percent of the yeast isolates were tolerant of temperatures up to 42 °C, 12.4% of them could tolerate ethanol concentrations up to 9% (v/v), 43.2% of them were tolerant to formic acid concentrations up to 20 mM, 22.7% were tolerant to acetic acid concentrations up to 45 mM, 34.0% of them could tolerate hydrogen peroxide up to 7 mM, and 44.3% of the yeasts could tolerate osmotic stress up to 1.5 M. CONCLUSION: The ability to tolerate multiple stresses is a desirable trait in the selection of novel production strains for diverse biotechnological applications, such as bioethanol production. Our study shows that the exploration of natural diversity in the search for stress-tolerant yeasts is an appealing approach for the development of robust yeasts.

Metabolic engineering strategies for naringenin production enhancement in Streptomyces albidoflavus J1074.

BACKGROUND: Naringenin is an industrially relevant compound due to its multiple pharmaceutical properties as well as its central role in flavonoid biosynthesis. RESULTS: On our way to develop Streptomyces albidoflavus J1074 as a microbial cell factory for naringenin production, we have significantly increased the yields of this flavanone by combining various metabolic engineering strategies, fermentation strategies and genome editing approaches in a stepwise manner. Specifically, we have screened different cultivation media to identify the optimal production conditions and have investigated how the additive feeding of naringenin precursors influences the production. Furthermore, we have employed genome editing strategies to remove biosynthetic gene clusters (BGCs) associated with pathways that might compete with naringenin biosynthesis for malonyl-CoA precursors. Moreover, we have expressed MatBC, coding for a malonate transporter and an enzyme responsible for the conversion of malonate into malonyl-CoA, respectively, and have duplicated the naringenin BGC, further contributing to the production improvement. By combining all of these strategies, we were able to achieve a remarkable 375-fold increase (from 0.06 mg/L to 22.47 mg/L) in naringenin titers. CONCLUSION: This work demonstrates the influence that fermentation conditions have over the final yield of a bioactive compound of interest and highlights various bottlenecks that affect production. Once such bottlenecks are identified, different strategies can be applied to overcome them, although the efficiencies of such strategies may vary and are difficult to predict.

Complementation of reducing power for 5-hydroxyvaleric acid and 1,5-pentanediol production via glucose dehydrogenase in Escherichia coli whole-cell system.

One of the key intermediates, 5-hydroxyvaleric acid (5-HV), is used in the synthesis of polyhydroxyalkanoate monomer, δ-valerolactone, 1,5-pentanediol (1,5-PDO), and many other substances. Due to global environmental problems, eco-friendly bio-based synthesis of various platform chemicals and key intermediates are socially required, but few previous studies on 5-HV biosynthesis have been conducted. To establish a sustainable bioprocess for 5-HV production, we introduced gabT encoding 4-aminobutyrate aminotransferase and yqhD encoding alcohol dehydrogenase to produce 5-HV from 5-aminovaleric acid (5-AVA), through glutarate semialdehyde in Escherichia coli whole-cell reaction. As, high reducing power is required to produce high concentrations of 5-HV, we newly introduced glucose dehydrogenase (GDH) for NADPH regeneration system from Bacillus subtilis 168. By applying GDH with D-glucose and optimizing the parameters, 5-HV conversion rate from 5-AVA increased from 47% (w/o GDH) to 82% when using 200 mM (23.4 g/L) of 5-AVA. Also, it reached 56% conversion in 2 h, showing 56 mM/h (6.547 g/L/h) productivity from 200 mM 5-AVA, finally reaching 350 mM (41 g/L) and 14.6 mM/h (1.708 g/L/h) productivity at 24 h when 1 M (117.15 g/L) 5-AVA was used. When the whole-cell system with GDH was expanded to produce 1,5-PDO, its production was also increased 5-fold. Considering that 5-HV and 1,5-PDO production depends heavily on the reducing power of the cells, we successfully achieved a significant increase in 5-HV and 1,5-PDO production using GDH.

A novel and economical approach for the synthesis of short rod-shaped mesoporous silica nanoparticles from coal fly ash waste by Bacillus circulans MTCC 6811.

Coal fly ash (CFA) is an industrial byproduct produced during the production of electricity in thermal power plants from the burning of pulverized coal. It is considered hazardous due to the presence of toxic heavy metals while it is also considered valuable due to the presence of value-added minerals like silicates, alumina, and iron oxides. Silica nanoparticles' demands and application have increased drastically in the last decade due to their mesoporous nature, high surface area to volume ratio, etc. Here in the present research work, short rod-shaped, mesoporous silica nanoparticles (MSN) have been synthesized from coal fly ash by using Bacillus circulans MTCC 6811 in two steps. Firstly, CFA was kept with the bacterial culture for bioleaching for 25 days in an incubator shaker at 120 rpm. Secondly, the dissolved silica in the medium was precipitated with the 4 M sodium hydroxide to obtain a short rod-shaped MSN. The purification of the synthesized silica particle was done by treating them with 1 M HCl at 120 °C, for 90 min. The synthesized short rod-shaped MSN were characterized by UV-vis spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Particle size analyzer (PSA), Field emission scanning electron microscopy (FESEM), and transmission electron microscope. The microscopic techniques revealed the short rod-shaped mesoporous silica nanoparticles (MSN) for the final nano-silica, whose size varies from 40 to 80 nm, with an average size of 36 ± 5 nm. The XRD shows the crystalline nature of the synthesized MSN having a crystallite size of 36 nm. The FTIR showed the three characteristic bands in the range of 400-1100 cm-1, indicating the purity of the sample. The energy dispersive X-ray (EDX) showed 53.04 wt% oxygen and 43.42% Si along with 3.54% carbon in the final MSN. The particle size analyzer revealed that the average particle size is 368.7 nm in radius and the polydispersity index (PDI) is 0.667. Such a novel and economical approach could be helpful in the synthesis of silica in high yield with high purity from coal fly ash and other similar waste.

Microbially Induced Calcium Carbonate Precipitation by Sporosarcina pasteurii: a Case Study in Optimizing Biological CaCO3 Precipitation.

Current production of traditional concrete requires enormous energy investment that accounts for approximately 5 to 8% of the world's annual CO2 production. Biocement is a building material that is already in industrial use and has the potential to rival traditional concrete as a more convenient and more environmentally friendly alternative. Biocement relies on biological structures (enzymes, cells, and/or cellular superstructures) to mineralize and bind particles in aggregate materials (e.g., sand and soil particles). Sporosarcina pasteurii is a workhorse organism for biocementation, but most research to date has focused on S. pasteurii as a building material rather than a biological system. In this review, we synthesize available materials science, microbiology, biochemistry, and cell biology evidence regarding biological CaCO3 precipitation and the role of microbes in microbially induced calcium carbonate precipitation (MICP) with a focus on S. pasteurii. Based on the available information, we provide a model that describes the molecular and cellular processes involved in converting feedstock material (urea and Ca2+) into cement. The model provides a foundational framework that we use to highlight particular targets for researchers as they proceed into optimizing the biology of MICP for biocement production.

Yeast population dynamics in Brazilian bioethanol production.

The large-scale and nonaseptic fermentation of sugarcane feedstocks into fuel ethanol in biorefineries represents a unique ecological niche, in which the yeast Saccharomyces cerevisiae is the predominant organism. Several factors, such as sugarcane variety, process design, and operating and weather conditions, make each of the ∼400 industrial units currently operating in Brazil a unique ecosystem. Here, we track yeast population dynamics in 2 different biorefineries through 2 production seasons (April to November of 2018 and 2019), using a novel statistical framework on a combination of metagenomic and clonal sequencing data. We find that variation from season to season in 1 biorefinery is small compared to the differences between the 2 units. In 1 biorefinery, all lineages present during the entire production period derive from 1 of the starter strains, while in the other, invading lineages took over the population and displaced the starter strain. However, despite the presence of invading lineages and the nonaseptic nature of the process, all yeast clones we isolated are phylogenetically related to other previously sequenced bioethanol yeast strains, indicating a common origin from this industrial niche. Despite the substantial changes observed in yeast populations through time in each biorefinery, key process indicators remained quite stable through both production seasons, suggesting that the process is robust to the details of these population dynamics.

Milligrams to kilograms: making microbes work at scale.

If biomanufacturing can become a sustainable route for producing chemicals, it will provide a critical step in reducing greenhouse gas emissions to fight climate change. However, efforts to industrialize microbial synthesis of chemicals have met with varied success, due, in part, to challenges in translating laboratory successes to industrial scale. With a particular focus on Escherichia coli, this review examines the lessons learned when studying microbial physiology and metabolism under conditions that simulate large-scale bioreactors and methods to minimize cellular waste through reduction of maintenance energy, optimizing the stress response and minimizing culture heterogeneity. With general strategies to overcome these challenges, biomanufacturing process scale-up could be de-risked and the time and cost of bringing promising syntheses to market could be reduced.

Enhancing the biomass and docosahexaenoic acid-rich lipid accumulation of Schizochytrium sp. in propionate wastewater.

In order to find a more effective way to obtain docosahexaenoic acid (DHA) rich lipid from Schizochytrium sp., a widespread propionate wastewater (PW) is used. PW is a common industrial and domestic wastewater, and transforming it into valuable products is a potential treatment method. Schizochytrium sp. is a rapidly growing oleaginous organism, which has been used commercially for DHA production. Herein, PW is completely used for DHA production by Schizochytrium sp. by genetic engineering and fermentation optimization, which can alleviate the increasingly tense demand for water resources and environmental pollution caused by industrial wastewater. Firstly, the methylmalonyl-CoA mutase (MCM) was overexpressed in Schizochytrium sp. to enhance the metabolism of propionate, then the engineered strain of overexpressed MCM (OMCM) can effectively use propionate. Then, the effects of PW with different concentration of propionate were investigated, and results showed that OMCM can completely replace clean water with PW containing 5 g L-1 propionate. Furthermore, in the fed-batch fermentation, the OMCM obtained the highest biomass of 113.4 g L-1 and lipid yield of 64.4 g L-1 in PW condition, which is 26.8% and 51.7% higher than that of wild type (WT) in PW condition. Moreover, to verify why overexpression of MCM can promote DHA and lipid accumulation, the comparative metabolomics, ATP production level, the antioxidant system, and the transcription of key genes were investigated. Results showed that ATP induced by PW condition could drive the synthesis of DHA, and remarkably improve the antioxidant capacity of cells by enhancing the carotenoids production. Therefore, PW can be used as an effective and economical substrate and water source for Schizochytrium sp. to accumulate biomass and DHA.

Recent advances in microbial production of medium chain fatty acid from renewable carbon resources: A comprehensive review.

Microbial production of medium chain length fatty acids (MCFAs) from renewable resources is becoming increasingly important in establishing a sustainable and clean chemical industry. This review comprehensively summarizes current advances in microbial MCFA production from renewable resources. Detailed information is provided on two major MCFA production pathways using various renewable resources and other auxiliary pathways supporting MCFA production to help understand the fundamentals of bio-based MCFA production. In addition, conventional and well-studied MCFA producers are classified into two categories, natural and synthetic producers, and their characteristics on MCFA production are outlined. Moreover, various engineering strategies employed to achieve the highest MCFAs production up to date are showcased together with key enzymes suggested for MCFA overproduction. Finally, future challenges and perspectives are discussed towards more efficient production of bio-based MCFA production.

Characterization of high-yield Bacillus subtilis cysteine protease for diverse industrial applications.

Bacterial proteases have extensive applications in various fields of industrial microbiology. In this study, protease-producing organisms were screened on skimmed milk agar media using serial dilution. Through microbial biomass production, biochemical tests, protease-specific activity, and 16 s RNA gene sequencing, the isolates were identified as Bacillus subtilis and submitted to NCBI. The strain accession numbers were designated as A1 (MT903972), A2 (MT903996), A4 (MT904091), and A5 (MT904796). The strain A4 Bacillus subtilis showed highest protease-specific activity as 76,153.84 U/mg. A4 Bacillus subtilis was unaffected by Ca2+, Cu2+, Fe2+, Hg2+, Mg2+, Na, Fe2+, and Zn2+ but was inhibited by 80% by Mn2+ (5 mM). The protease activity was inhibited by up to 30% by iodoacetamide (5 mM). These findings confirm the enzyme to be a cysteine protease which was further confirmed by MALDI-TOF. The identified protease showed 71% sequence similarity with Bacillus subtilis cysteine protease. The crude cysteine protease significantly aided in fabric stain removal when added to a generic detergent. It also aided in the recovery of silver from used X-ray films and de-hairing of goat skin hides and showed decent application in meat tenderization. Thus, the isolated cysteine protease has high potential for industrial applications.

Deletion of QDR genes in a bioethanol-producing yeast strain reduces propagation of contaminating lactic acid bacteria.

Bacterial contaminations in yeast fermentation tanks are a recurring problem for the bioethanol production industry. Lactic acid bacteria (LAB), particularly of the genus Lactobacillus, are the most common contaminants. Their proliferation can reduce fermentation efficiency or even impose premature shutdown for cleaning. We have previously reported that laboratory yeast strains naturally excrete amino acids via transporters of the Drug: H+ Antiporter-1 (DHA1) family. This excretion allows yeast to cross-feed LAB, which are most often unable to grow without an external amino acid supply. Whether industrial yeast strains used in bioethanol production likewise promote LAB proliferation through cross-feeding has not been investigated. In this study, we first show that the yeast strain Ethanol Red used in ethanol production supports growth of Lactobacillus fermentum in an amino-acid-free synthetic medium. This effect was markedly reduced upon homozygous deletion of the QDR3 gene encoding a DHA1-family amino acid exporter. We further show that cultivation of Ethanol Red in a nonsterile sugarcane-molasses-based medium is associated with an increase in lactic acid due to LAB growth. When Ethanol Red lacked the QDR1, QDR2, and QDR3 genes, this lactic acid production was not observed and ethanol production was not significantly reduced. Our results indicate that Ethanol Red cultivated in synthetic or molasses medium sustains LAB proliferation in a manner that depends on its ability to excrete amino acids via Qdr transporters. They further suggest that using mutant industrial yeast derivatives lacking DHA1-family amino acid exporters may be a way to reduce the risk of bacterial contaminations during fermentation.

[Adaptive evolution of microorganisms based on industrial environmental perturbations].

The development of synthetic biology has greatly promoted the construction of microbial cell factories, providing an important strategy for green and efficient chemical production. However, the bottleneck of poor tolerance to harsh industrial environments has become the key factor hampering the productivity of microbial cells. Adaptive evolution is an important method to domesticate microorganisms for a certain period by applying targeted selection pressure to obtain desired phenotypic or physiological properties that are adapted to a specific environment. Recently, with the development of technologies such as microfluidics, biosensors, and omics analysis, adaptive evolution has laid the foundation for efficient productivity of microbial cell factories. Herein, we discuss the key technologies of adaptive evolution and their important applications in improvement of environmental tolerance and production efficiency of microbial cell factories. Moreover, we looked forward to the prospects of adaptive evolution to realize industrial production by microbial cell factories.

Weaponising microbes for peace.

There is much human disadvantage and unmet need in the world, including deficits in basic resources and services considered to be human rights, such as drinking water, sanitation and hygiene, healthy nutrition, access to basic healthcare, and a clean environment. Furthermore, there are substantive asymmetries in the distribution of key resources among peoples. These deficits and asymmetries can lead to local and regional crises among peoples competing for limited resources, which, in turn, can become sources of discontent and conflict. Such conflicts have the potential to escalate into regional wars and even lead to global instability. Ergo: in addition to moral and ethical imperatives to level up, to ensure that all peoples have basic resources and services essential for healthy living and to reduce inequalities, all nations have a self-interest to pursue with determination all available avenues to promote peace through reducing sources of conflicts in the world. Microorganisms and pertinent microbial technologies have unique and exceptional abilities to provide, or contribute to the provision of, basic resources and services that are lacking in many parts of the world, and thereby address key deficits that might constitute sources of conflict. However, the deployment of such technologies to this end is seriously underexploited. Here, we highlight some of the key available and emerging technologies that demand greater consideration and exploitation in endeavours to eliminate unnecessary deprivations, enable healthy lives of all and remove preventable grounds for competition over limited resources that can escalate into conflicts in the world. We exhort central actors: microbiologists, funding agencies and philanthropic organisations, politicians worldwide and international governmental and non-governmental organisations, to engage - in full partnership - with all relevant stakeholders, to 'weaponise' microbes and microbial technologies to fight resource deficits and asymmetries, in particular among the most vulnerable populations, and thereby create humanitarian conditions more conducive to harmony and peace.

Bacillus spp. as Bio-factories for Antifungal Secondary Metabolites: Innovation Beyond Whole Organism Formulations.

Several fungi act as parasites for crops causing huge annual crop losses at both pre- and post-harvest stages. For years, chemical fungicides were the solution; however, their wide use has caused environmental contamination and human health problems. For this reason, the use of biofungicides has been in practice as a green solution against fungal phytopathogens. In the context of a more sustainable agriculture, microbial biofungicides have the largest share among the commercial biocontrol products that are available in the market. Precisely, the genus Bacillus has been largely studied for the management of plant pathogenic fungi because they offer a chemically diverse arsenal of antifungal secondary metabolites, which have spawned a heightened industrial engrossment of it as a biopesticide. In this sense, it is indispensable to know the wide arsenal that Bacillus genus has to apply these products for sustainable agriculture. Having this idea in our minds, in this review, secondary metabolites from Bacillus having antifungal activity are chemically and structurally described giving details of their action against several phytopathogens. Knowing the current status of Bacillus secreted antifungals is the base for the goal to apply these in agriculture and it is addressed in depth in the second part of this review.

Crucial aspects of metabolism and cell biology relating to industrial production and processing of Saccharomyces biomass.

The multitude of applications to which Saccharomyces spp. are put makes these yeasts the most prolific of industrial microorganisms. This review considers biological aspects pertaining to the manufacture of industrial yeast biomass. It is proposed that the production of yeast biomass can be considered in two distinct but interdependent phases. Firstly, there is a cell replication phase that involves reproduction of cells by their transitions through multiple budding and metabolic cycles. Secondly, there needs to be a cell conditioning phase that enables the accrued biomass to withstand the physicochemical challenges associated with downstream processing and storage. The production of yeast biomass is not simply a case of providing sugar, nutrients, and other growth conditions to enable multiple budding cycles to occur. In the latter stages of culturing, it is important that all cells are induced to complete their current budding cycle and subsequently enter into a quiescent state engendering robustness. Both the cell replication and conditioning phases need to be optimized and considered in concert to ensure good biomass production economics, and optimum performance of industrial yeasts in food and fermentation applications. Key features of metabolism and cell biology affecting replication and conditioning of industrial Saccharomyces are presented. Alternatives for growth substrates are discussed, along with the challenges and prospects associated with defining the genetic bases of industrially important phenotypes, and the generation of new yeast strains."I must be cruel only to be kind: Thus bad begins, and worse remains behind." William Shakespeare: Hamlet, Act 3, Scene 4.

Bioeconomy and net-zero carbon: lessons from Trends in Biotechnology, volume 1, issue 1.

Many biotechnology applications tend to be for low production volumes and relatively high-value products such as insulin and vaccines. More difficult to perfect at scale are bioprocesses for high-volume products with lower value, especially if the target product is a reduced chemical such as a solvent or a plastic. Historically, industrial microbiology succeeded under special circumstances when fossil feedstocks were either unavailable or expensive. Inevitably, as these circumstances relaxed, bioprocesses struggled to compete with petrochemistry. Why try to compete? Fossil resources will be phased out in the coming decades in the struggle with climate change. To reach net-zero carbon by 2050 will require all sectors to transition, not only energy and transportation. This may herald a new opportunity for industrial bioprocesses with much better tools.

Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates / Produção significativa de vanilina e amplificação in vitro do gene ech em isolados bacterianos locais

Abstract 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.

Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.