Process model economics of xanthan production from confectionery industry wastewaters.

In this research a process and cost model for a xanthan production facility was developed using process simulation software (SuperPro Designer®). This work represents a novelty in the field for two reasons. One is that xanthan gum has been produced from several wastes but never from wastewaters from confectionery industries. The other more important is that the aforementioned software, which in intended exclusively for bioprocesses, is used for generating a base case, i.e. starting point for transferring the technology to industrial scales. Previously acquired experimental knowledge about using confectionery wastewaters from five different factories as substitutes for commercially used cultivation medium have been incorporated into the process model in order to obtain an economic viability of implementing such substrates. A lower initial sugar content in the medium based on wastewater (28.41 g/L) compared to the synthetic medium (30.00 g/L) gave a lower xanthan content at the end of cultivation (23.98 and 26.27 g/L, respectively). Although this resulted in somewhat poorer economic parameters, they were still in the range of being an investment of interest. Also the possibility of utilizing a cheap resource (waste) and reducing pollution that would result from its disposal has a positive effect on the environment.

Bacillus VOCs in the Context of Biological Control.

A contemporary agricultural production system relying on heavy usage of agrochemicals represents a questionable outlook for sustainable food supply in the future. The visible negative environmental impacts and unforeseen consequences to human and animal health have been requiring a shift towards the novel eco-friendly alternatives for chemical pesticides for a while now. Microbial-based biocontrol agents have shown a promising potential for plant disease management. The bacteria of the genus Bacillus have been among the most exploited microbial active components due to several highly efficient mechanisms of action against plant pathogens, as well as a palette of additional plant-beneficial mechanisms, together with their suitable properties for microbial biopesticide formulations. Among other bioactive metabolites, volatile organic compounds (VOCs) have been investigated for their biocontrol applications, exhibiting the main advantage of long-distance effect without the necessity for direct contact with plants or pathogens. The aim of this study is to give an overview of the state-of-the-art in the field of Bacillus-based VOCs, especially in terms of their antibacterial, antifungal, and nematicidal action as the main segments determining their potential for biocontrol applications in sustainable agriculture.

Bacillus-Loaded Biochar as Soil Amendment for Improved Germination of Maize Seeds.

Biochar is considered one of the most promising long-term solutions for soil quality improvement, representing an ideal environment for microorganisms' immobilization. Hence there is a possibility to design microbial products formulated using biochar as a solid carrier. The present study was aimed at development and characterization of Bacillus-loaded biochar to be applied as a soil amendment. The producing microorganism Bacillus sp. BioSol021 was evaluated in terms of plant growth promotion traits, indicating significant potential for production of hydrolytic enzymes, indole acetic acid (IAA) and surfactin and positive tests for ammonia and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production. Soybean biochar was characterised in terms of physicochemical properties to evaluate its suitability for agricultural applications. The experimental plan for Bacillus sp. BioSol021 immobilisation to biochar included variation of biochar concentration in cultivation broth and adhesion time, while the soil amendment effectiveness was evaluated during maize germination. The best results in terms of maize seed germination and seedling growth promotion were achieved by applying 5% of biochar during the 48 h immobilisation procedure. Germination percentage, root and shoot length and seed vigour index were significantly improved when using Bacillus-biochar soil amendment compared to separate treatments including biochar and Bacillus sp. BioSol021 cultivation broth. The results indicated the synergistic effect of producing microorganism and biochar on maize seed germination and seedling growth promotion, pointing out the promising potential of this proposed multi-beneficial solution for application in agricultural practices.

Medium for the Production of Bacillus-Based Biocontrol Agent Effective against Aflatoxigenic Aspergillus flavus: Dual Approach for Modelling and Optimization.

One of the leading limiting factors for wider industrial production and commercialization of microbial biopesticides refers to the high costs of cultivation media. The selection of alternative sources of macronutrients crucial for the growth and metabolic activity of the producing microorganism is a necessary phase of the bioprocess development. Gaining a better understanding of the influence of the medium composition on the biotechnological production of biocontrol agents is enabled through bioprocess modelling and optimization. In the present study, after the selection of optimal carbon and nitrogen sources, two modelling approaches were applied to mathematically describe the behavior of the examined bioprocess-the production of biocontrol agents effective against aflatoxigenic Aspergillus flavus strains. The modelling was performed using four independent variables: cellulose, urea, ammonium sulfate and dipotassium phosphate, and the selected response was the inhibition-zone diameter. After the comparison of the results generated by the Response Surface Methodology (RSM) and the Artificial Neural Network (ANN) approach, the first model was chosen for the further optimization step due to the better fit of the experimental results. As the final investigation step, the optimal cultivation medium composition was defined (g/L): cellulose 5.0, ammonium sulfate 3.77, dipotassium phosphate 0.3, magnesium sulfate heptahydrate 0.3.

Kenics Static Mixer Combined with Gas Sparging for the Improvement of Cross-Flow Microfiltration: Modeling and Optimization.

The use of membrane filtration as a downstream process for microbial biomass harvesting is hampered due to the low permeate flux values achieved during the microfiltration of fermentation broths. Several hydrodynamic methods for increasing permeate flux by creating turbulent flow patterns inside the membrane module are used to overcome this problem. The main goal of this study was to investigate the combined use of a Kenics static mixer and gas sparging during cross-flow microfiltration of Bacillus velezensis IP22 cultivation broth. Optimization of the microfiltration process was performed by using the response surface methodology. It was found that the combined use of a static mixer and gas sparging leads to a considerable increase in the permeate flux, up to the optimum steady-state permeate flux value of 183.42 L·m-2·h-1 and specific energy consumption of 0.844 kW·h·m-3. The optimum steady-state permeate flux is almost four times higher, whilst, at the same time, the specific energy consumption is almost three times lower compared to the optimum results achieved using gas sparging alone. The combination of Kenics static mixer and gas sparging during cross-flow microfiltration is a promising technique for the enhancement of steady-state permeate flux with simultaneously decreasing specific energy consumption.

Best-performing Bacillus strains for microbiologically induced CaCO3 precipitation: Screening of relative influence of operational and environmental factors.

The microbiologically induced calcite precipitation (MICP) can be an emerging approach that could tap onto soil bacterial diversity and use as a bioremediation technique. Based on the concept that bacteria with biomineralization capacity could be effective CaCO3 inductance agents, this study aimed to evaluate the simultaneous influence of 11 operational and environmental factors on the MICP process, for the first time. Therefore, Bacillus muralis, B. lentus, B. simplex, B. firmus, and B. licheniformis, isolated from alkaline soils, were used in the selection of the best performing bacterium compared with a well-known MICP bioagent Sporosarcina pasteurii DSM 33. Plackett-Burman's experimental design was labouring to screen all independent variables for their significances on five outputs (pH value, number of viable cells and spores, amount of urea and CaCO3 precipitate). According to experimentally obtained data, an artificial neural network model based on the Broyden-Fletcher-Goldfarb-Shanno algorithm showed good prediction capabilities, while differences in the relative influences were observed at the bacterial strain level. B. licheniformis turn out to be the most potent bioagent, with a maximum amount of CaCO3 precipitate of 3.14 g/100 mL in the optimal conditions.

Dairy and Wine Industry Effluents as Alternative Media for the Production of Bacillus-Based Biocontrol Agents.

Food industry effluents represent one of the major concerns when it comes to environmental impact; hence, their valorization through different chemical and biological routes has been suggested as a possible solution. The vast amount of organic and inorganic nutrients present in food industry effluents makes them suitable substrates for microbial growth. This study suggests two valorization routes for whey as dairy industry effluent and flotation wastewater from the wine industry through microbial conversion to biocontrol agents as value-added products. Cultivations of the biocontrol strain Bacillus sp. BioSol021 were performed in a 16 L bioreactor to monitor the bioprocess course and investigate bioprocess kinetics in terms of microbial growth, sugar substrate consumption and surfactin synthesis, as an antimicrobial lipopeptide. The produced biocontrol agents showed high levels of biocontrol activity against mycotoxigenic strains of Aspergillus flavus, followed by a significant reduction of sugar load of the investigated effluents by the producing microorganisms. With proven high potential of whey and winery flotation wastewater to be used as substrates for microbial growth, this study provides grounds for further optimization of the suggested valorization routes, mostly in terms of bioprocess conditions to achieve maximal techno-economical feasibility, energy saving and maximal reduction of effluents' organic and inorganic burden.

Utilization of waste glycerol for the production of biocontrol agents nigericin and niphimycin by Streptomyces hygroscopicus: bioprocess development.

Search for more environment-friendly methods for controlling plant diseases that would contribute to the goal of sustainability in agriculture is in focus. In the present study, the potential of Streptomyces hygroscopicus isolated from soil sample in the production of biocontrol agents, nigericin and niphimycin, effective against Alternaria alternata storage apple pathogen was examined. Also, modelling and optimization of medium composition for biocontrol agent biosynthesis was performed. The results showed that the optimum amount of C3H8O3, (NH4)2SO4 and K2HPO4 in the medium for Streptomyces hygroscopicus biosynthesis is 20, 0.25 and 1.46 g/L, respectively. Scale-up and validation of the obtained results performed in the 3 L laboratory-scale bioreactor showed that on the optimized medium at an aeration rate of 0.7 vvm and an agitation speed of 200 rpm, produced nigericin and niphimycin, showed high activity. Under the same conditions, cultivation of S. hygroscopicus was performed in a 7 L laboratory bioreactor in a medium with waste glycerol instead of pure glycerol. Results showed that the methanol extract of S. hygroscopicus cultivation liquid, containing nigericin and niphimycin, was high effective against two Alternaria isolates. This was confirmed in vitro by obtaining large inhibition zone diameters on A. alternata KA10 (47 mm) and T1Jg3 (44.33 mm) isolates. After successful in vitro analysis, in planta testing was performed. It was found that necrosis diameters that were measured on artificially inoculated apple fruits with A. alternata compared to necrosis diameter measured on untreated, control fruits, were 4.47 and 3.56 times smaller.

Taguchi Grey Relational Analysis for Multi-Response Optimization of Bacillus Bacteria Flocculation Recovery from Fermented Broth by Chitosan to Enhance Biocontrol Efficiency.

Degradation of environment is a challenge to crop production around the world. Biological control of various plant diseases using antagonistic bacteria is an encouraging alternative to traditionally used chemical control strategies. Chitosan as a well-known natural flocculation agent also exhibits antimicrobial activity. The goal of this study was to investigate a dual nature of chitosan in flocculation of Bacillus sp. BioSol021 cultivation broth intended for biocontrol applications. Experiments were performed based on L18 standard Taguchi orthogonal array design with five input parameters (chitosan type and dosage, pH value, rapid and slow mixing rates). In this study, the grey relational analysis was used to perform multi-objective optimization of the chosen responses, i.e., flocculation efficiency and four inhibition zone diameters against the selected phytopathogens. The results have indicated a great potential of a highly efficient method for removal of the Bacillus bacteria from the cultivation broth using chitosan. The good flocculation efficiency and high precipitate antimicrobial activity against the selected phytopathogens were achieved. It has been shown that multiple flocculation performance parameters were improved, resulting in slightly improved response values.

The Effect of Cultivation Conditions on Antifungal and Maize Seed Germination Activity of Bacillus-Based Biocontrol Agent.

Aflatoxin contamination is a global risk and a concerning problem threatening food safety. The biotechnological answer lies in the production of biocontrol agents that are effective against aflatoxins producers. In addition to their biocontrol effect, microbial-based products are recognized as efficient biosolutions for plant nutrition and growth promotion. The present study addresses the characterization of the representative of Phaseolus vulgaris rhizosphere microbiome, Bacillus sp. BioSol021, regarding plant growth promotion traits, including the activity of protease, cellulase, xylanase, and pectinase with the enzymatic activity index values 1.06, 2.04, 2.41, and 3.51, respectively. The potential for the wider commercialization of this kind of product is determined by the possibility of developing a scalable bioprocess solution suitable for technology transfer to an industrial scale. Therefore, the study addresses one of the most challenging steps in bioprocess development, including the production scale-up from the Erlenmeyer flask to the laboratory bioreactor. The results indicated the influence of the key bioprocess parameters on the dual mechanism of action of biocontrol effects against the aflatoxigenic Aspergillus flavus, as well on maize seed germination activity, pointing out the positive impact of high aeration intensity and agitation rate, resulting in inhibition zone diameters of 60 mm, a root length 96 mm, and a shoot length 27 mm.

Genetic Diversity of Pectobacterium spp. on Potato in Serbia.

Pectobacterium is a diverse genus which comprises of multiple destructive bacterial species which cause soft rot/blackleg/wilt disease complex in a wide variety of crops by employing high levels of virulence factors. During the 2018, 2019 and 2020 potato growing seasons, numerous outbreaks of bacterial wilt, stem blackleg and tuber soft rot were recorded, and symptomatic plant samples from ten localities in the Province of Vojvodina (Serbia) were collected and analysed. Bacterial soft-rot pathogens were detected in 63 samples using genus and species-specific primers. Through 16S rRNA Sanger sequencing of 19 representative isolates, the identity of P. brasiliense (73.7%), P. punjabense (15.8%), and P. carotovorum (10.5%) species were revealed. To further validate the identification, genotypic profiling of Pectobacterium strains using rep-PCR (ERIC, BOX, REP) was conducted for 25 selected isolates and the phylogenetic assessment based on four selected housekeeping genes (gyrA, recA, rpoA, and rpoS). Physiological and biochemical properties were analysed using basic microbiological tests and VITEK® 2 GN card, and pathogenicity was confirmed on cv. VR808 and cv. Desiree potato tubers and plants. This study confirmed the distinctiveness of the newly described P. punjabense in Serbia as well as the high diversity of Pectobacterium brasiliense and Pectobacterium carotovorum species in Serbia.

Bioengineering Outlook on Cultivated Meat Production.

Cultured meat (also referred to as cultivated meat or cell-based meat)-CM-is fabricated through the process of cellular agriculture (CA), which entails application of bioengineering, i.e., tissue engineering (TE) principles to the production of food. The main TE principles include usage of cells, grown in a controlled environment provided by bioreactors and cultivation media supplemented with growth factors and other needed nutrients and signaling molecules, and seeded onto the immobilization elements-microcarriers and scaffolds that provide the adhesion surfaces necessary for anchor-dependent cells and offer 3D organization for multiple cell types. Theoretically, many solutions from regenerative medicine and biomedical engineering can be applied in CM-TE, i.e., CA. However, in practice, there are a number of specificities regarding fabrication of a CM product that needs to fulfill not only the majority of functional criteria of muscle and fat TE, but also has to possess the sensory and nutritional qualities of a traditional food component, i.e., the meat it aims to replace. This is the reason that bioengineering aimed at CM production needs to be regarded as a specific scientific discipline of a multidisciplinary nature, integrating principles from biomedical engineering as well as from food manufacturing, design and development, i.e., food engineering. An important requirement is also the need to use as little as possible of animal-derived components in the whole CM bioprocess. In this review, we aim to present the current knowledge on different bioengineering aspects, pertinent to different current scientific disciplines but all relevant for CM engineering, relevant for muscle TE, including different cell sources, bioreactor types, media requirements, bioprocess monitoring and kinetics and their modifications for use in CA, all in view of their potential for efficient CM bioprocess scale-up. We believe such a review will offer a good overview of different bioengineering strategies for CM production and will be useful to a range of interested stakeholders, from students just entering the CA field to experienced researchers looking for the latest innovations in the field.

Modeling and Optimization of Gas Sparging-Assisted Bacterial Cultivation Broth Microfiltration by Response Surface Methodology and Genetic Algorithm.

Production of highly efficient biomass-based microbial biopesticides significantly depends on downstream processing in terms of obtaining as high concentration of viable cells as possible. Microfiltration is one of the recommended operations for microbial biomass separation, but its main limitation is permeate flux decrease due to the membrane fouling. The effect of air sparging as a hydrodynamic technique for improvement of permeate flux during microfiltration of Bacillus velezensis cultivation broth was investigated. Modeling of the microfiltration was performed using the response surface methodology, while desirability function approach and genetic algorithm were applied for optimization, i.e., maximization of permeate flux and minimization of specific energy consumption. The results have revealed antagonistic relationship between the investigated dependent variables. The optimized values of superficial feed velocity and transmembrane pressure were close to the mean values of the investigated value ranges (0.68 bar and 0.96 m/s, respectively), while the optimized value of superficial air velocity had a more narrow distribution around 0.25 m/s. The results of this study have revealed a significant improvement of microfiltration performance by applying air sparging, thus this flux improvement method should be further investigated in downstream processing of different bacterial cultivation broths.

Distribution, Genetic Diversity and Biocontrol of Aflatoxigenic Aspergillus flavus in Serbian Maize Fields.

Maize is one of the leading export products in the Republic of Serbia. As a country where economic development depends on agriculture, maize production plays a critical role as a crop of strategic importance. Potential aflatoxin contamination of maize poses a risk to food and feed safety and tremendous economic losses. No aflatoxin contamination of maize samples harvested in 2019 and 2020 in different localities in the Republic of Serbia was detected by the Enzyme-Linked Immunosorbent Assay (ELISA) test and High-Performance Liquid Chromatography (HPLC) method. On the other hand, the Cluster Amplification Patterns (CAP) analyses of the isolated Aspergillus flavus strains from 2019 maize samples confirmed the presence of key biosynthesis genes responsible for aflatoxin production. Artificial inoculation and subsequent HPLC analysis of the inoculated maize samples confirmed the high capacity of the A. flavus strains for aflatoxin production, pointing to a high risk of contamination under favorable conditions. Prevention of aflatoxin contamination is primarily based on A. flavus control, where biocontrol agents play a significant role as sustainable disease management tools. In this study, antagonistic activity screening of the novel strains belonging to the Bacillus genus indicated superior suppression of A. flavus strains by two Bacillus strains isolated from the rhizosphere of Phaseolus vulgaris.

Pepper Bacterial Spot Control by Bacillus velezensis: Bioprocess Solution.

Pepper bacterial spot is one of the most severe plant diseases in terms of infection persistence and economic losses when it comes to fresh pepper fruits used in nutrition and industrial processing. In this study, Bacillus velezensis IP22 isolated from fresh cheese was used as a biocontrol agent of pepper bacterial spot, whose main causal agent is the cosmopolitan pathogen Xanthomonas euvesicatoria. After optimization of the cultivation medium composition aimed at maximizing of the antimicrobial activity against X. euvesicatoria and validation of the optimized medium at the scale of a laboratory bioreactor, in planta tests were performed. The results have showed significant suppression of bacterial spot symptoms in pepper plants by the produced biocontrol agent, as well as reduction of disease spreading on the healthy (uninoculated) pepper leaves. Furthermore, HPLC-MS (high pressure liquid chromatography-mass spectrometry) analysis was employed to examine antimicrobial metabolites produced by B. velezensis IP22, where lipopeptides were found with similar m/z values compared to lipopeptides from fengycin and locillomycin families. The bioprocess solution developed at the laboratory scale investigated in this study represents a promising strategy for production of pepper bacterial spot biocontrol agent based on B. velezensis IP22, a food isolate with a great perspective for application in plant protection.

Dynamic Modeling Using Artificial Neural Network of Bacillus Velezensis Broth Cross-Flow Microfiltration Enhanced by Air-Sparging and Turbulence Promoter.

Cross-flow microfiltration is a broadly accepted technique for separation of microbial biomass after the cultivation process. However, membrane fouling emerges as the main problem affecting permeate flux decline and separation process efficiency. Hydrodynamic methods, such as turbulence promoters and air sparging, were tested to improve permeate flux during microfiltration. In this study, a non-recurrent feed-forward artificial neural network (ANN) with one hidden layer was examined as a tool for microfiltration modeling using Bacillus velezensis cultivation broth as the feed mixture, while the Kenics static mixer and two-phase flow, as well as their combination, were used to improve permeate flux in microfiltration experiments. The results of this study have confirmed successful application of the ANN model for prediction of permeate flux during microfiltration of Bacillus velezensis cultivation broth with a coefficient of determination of 99.23% and absolute relative error less than 20% for over 95% of the predicted data. The optimal ANN topology was 5-13-1, trained by the Levenberg-Marquardt training algorithm and with hyperbolic sigmoid transfer function between the input and the hidden layer.

Optimization of glycerol-based medium composition for antifungal metabolites production by Bacillus subtilis

Abstract Bacillus species are promising producers of various compounds that have pronounced antimicrobial, antiviral and antitumor activities. Due to its GRAS status, Bacillus subtilis represents an excellent candidate for the usage in plant pathogens biocontrol. In this research, evaluation of antifungal metabolites biosynthesis by Bacillus subtilis ATCC 6633 and optimization of glycerol-based medium composition, using response surface methodology, for the production of compounds effective against Neurospora crassa were investigated. The results of disc-diffusion method indicate that applied Bacillus strain produces compounds with antifungal activity against tested fungus. In order to find optimal cultivation medium composition, the experiments were carried out in accordance with Box-Behnken design, and optimization was performed using the concept of desirability function combined with previously defined mathematical equation, which describes examined bioprocess. The optimization model predicts that maximum inhibition zone diameter against Neurospora crassa of 32.24 mm is achieved when initial content of glycerol, NaNO2 and K2HPO4 were 49.68 g/L, 2.90 g/L and 6.49 g/L, respectively. Additionally, the second optimization set was made to minimize the consumption of medium components and costs of medium preparation. The obtained results are the basis for further research aimed to develop medium appropriate for economically justified production of bioactive compounds at industrial scale.

Chemometric approach to prediction of antibacterial agent production by Streptomyces hygroscopicus.

The nutritional requirements for antimicrobial agent production using Streptomyces hygroscopicus were analyzed in shake flask experiments. Antimicrobial activity was tested against Staphylococcus aureus and Bacillus cereus. The mathematical models have been generated with relative high complexity in order to give an adequate fit to the data. All the results suggest a high dependence of produced antimicrobial agent quantities on the amount of carbon, nitrogen, and phosphorus in cultivation medium. The statistical results of the generated models reflect the high predictive ability. The derived models were validated using leave-one-out cross-validation technique, and from statistical point of view, they have significantly high values of the cross-validation parameters.

Process model and economic analysis of ethanol production from sugar beet raw juice as part of the cleaner production concept.

The batch fermentation process of sugar beet processing intermediates by free yeast cells is the most widely used method in the Autonomous Province of Vojvodina for producing ethanol as fuel. In this study a process and cost model was developed for producing ethanol from raw juice. The model can be used to calculate capital investment costs, unit production costs and operating costs for a plant producing 44 million l of 99.6% pure ethanol annually. In the sensitivity analysis the influence of sugar beet and yeast price, as well as the influence of recycled biomass on process economics, ethanol production costs and project feasibility was examined. The results of this study clearly demonstrate that the raw material costs have a significant influence on the expenses for producing ethanol. Also, the optimal percentage of recycled biomass turned out to be in the range from 50% to 70%.