Plant growth-promoting and biocontrol traits of endophytic Bacillus licheniformis against soft rot causing Pythium myriotylum in ginger plant.

Bacterial endophytes from plants harbor diverse metabolites that play major roles in biocontrol and improve plant growth. In this study, a total of 12 endophytic bacteria were isolated from the ginger rhizome. The strain K3 was highly effective in preventing mycelia growth of Pythium myriotylum (78.5 ± 1.5% inhibition) in dual culture. The cell-free extract (2.5%) of endophyte K3 inhibited 76.3 ± 4.8% mycelia growth, and 92.4 ± 4.2% inhibition was observed at a 5% sample concentration. The secondary metabolites produced by Bacillus licheniformis K3 showed maximum activity against Pseudomonas syringae (24 ± 1 mm zone of inhibition) and Xanthomonas campestris (28 ± 3 mm zone of inhibition). The strain K3 produced 28.3 ± 1.7 IU mL-1 protease, 28.3 ± 1.7 IU mL-1 cellulase, and 2.04 ± 0.13 IU mL-1 chitinase, respectively. The ginger rhizome treated with K3 in the greenhouse registered 53.8 ± 1.4% soft rot incidence, and the streptomycin-treated pot registered 78.3 ± 1.7% disease incidence. The selected endophyte K3 improved ascorbate peroxidase (1.37 ± 0.009 µmole ASC min-1 mg-1 protein), catalase (8.7 ± 0.28 µmole min-1 mg-1 protein), and phenylalanine ammonia-lyase (26.2 ± 0.99 Umg-1) in the greenhouse. In addition, K3 treatment in the field trial improved rhizome yield (730 ± 18.4 g) after 180 days (p < 0.01). The shoot length was 46 ± 8.3 cm in K3-treated plants, and it was about 31% higher than the control treatment (p < 0.01). The lytic enzyme-producing and growth-promoting endophyte is useful in sustainable crop production through the management of biotic stress.

Detection of risk areas in dairy powder processes: The development of thermophilic spore forming bacteria taking into account their growth limits.

Anoxybacillus flavithermus, Geobacillus stearothermophilus and Bacillus licheniformis are the main contaminants found in dairy powders. These spore-forming thermophilic bacteria, rarely detected in raw milk, persist, and grow during the milk powder manufacturing process. Moreover, in the form of spores, these species resist and concentrate in the powders during the processes. The aim of this study was to determine the stages of the dairy powder manufacturing processes that are favorable to the growth of such contaminants. A total of 5 strains were selected for each species as a natural contaminant of dairy pipelines in order to determine the minimum and maximum growth enabling values for temperature, pH, and aw and their optimum growth rates in milk. These growth limits were combined with the environmental conditions of temperature, pH and aw encountered at each step of the manufacture of whole milk, skim milk and milk protein concentrate powders to estimate growth capacities using cardinal models and the Gamma concept. These simulations were used to theoretically calculate the population sizes reached for the different strains studied at each stage in between two successive cleaning in place procedures. This approach highlights the stages at which risk occurs for the development of spore-forming thermophilic bacterial species. During the first stages of production, i.e. pre-treatment, pasteurization, standardization and pre-heating before concentration, physico-chemical conditions encountered are suitable for the development and growth of A. flavithermus, G. stearothermophilus and B. licheniformis. During the pre-heating stage and during the first effects in the evaporators, the temperature conditions appear to be the most favorable for the growth of G. stearothermophilus. The temperatures in the evaporator during the last evaporator effects are favorable for the growth of B. licheniformis. In the evaporation stage, low water activity severely limits the development of A. flavithermus.

Effect of a novel alkaline protease from Bacillus licheniformis on growth performance, carcass characteristics, meat quality, antioxidant capacity, and intestinal morphology of white feather broilers.

BACKGROUND: The present study was conducted to investigate the effects of dietary novel alkaline protease from Bacillus licheniformis on the growth performance, meat quality, antioxidant status and intestinal morphology of broilers. In total, 4000 broilers were randomly assigned into five groups and treated with normal control, normal control + 100 mg kg-1 protease, normal control + 200 mg kg-1 protease, normal control + 300 mg kg-1 protease and normal control + 400 mg kg-1 protease. RESULTS: Supplementing protease impacted final body weight (linear, P = 0.003; quadratic, P = 0.006) and decreased feed conversion rate (linear, P = 0.036) in broilers. Moreover, dietary protease significantly increased breast muscle rate (linear, P = 0.005; quadratic, P = 0.021) and decreased drip loss (linear, P < 0.001; quadratic, P < 0.001). In addition, dietary protease notably increased protein digestibility (linear, P = 0.001; quadratic, P = 0.006) and trypsin activity (linear, P = 0.002; quadratic, P = 0.009) in jejunum. Light microscopy revealed that the jejunum villi in the 300 mg kg-1 and 400 mg kg-1 groups exhibited greater height and a denser arrangement compared to those in the control group. The addition of protease decreased malondialdehyde content (linear, P < 0.001; quadratic, P < 0.001) and increased total antioxidant capacity (linear, P = 0.001; quadratic, P < 0.001) in pectoral muscles. CONCLUSION: The results of the present study suggest that dietary novel alkaline protease from B. licheniformis improved growth performance by affecting trypsin activity, protein digestibility, antioxidant capacity and intestinal health. © 2024 Society of Chemical Industry.

Poly-γ-glutamic acid overproduction of Bacillus licheniformis ATCC 9945a by developing a novel optimum culture medium and glutamate pulse feeding using different experimental design approaches.

The commercial production of multifunctional, biocompatible, and biodegradable biopolymers such as poly-γ-glutamic acid via microbial fermentation requires the development of simple and cheap methods for mass production. This study optimized the poly-γ-glutamic acid production of Bacillus licheniformis ATCC 9945a in several steps. At first, the most critical components of the culture medium, including l-glutamic acid, citric acid, and glycerol, were selected by screening nine factors through the Plackett-Burman experimental design and then were optimized using the response surface method and the central composite design algorithm. Under optimal conditions, the production of poly-γ-glutamic acid increased by more than 4.2 times from 11.2 to 47.2 g/L. This is one of the highest production rates of this strain in submerged batch fermentation reported so far using the optimized medium compared to the conventional base medium. A novel and efficient sudden pulse feeding strategy (achieved by a novel one-factorial statistical technique) of l-glutamic acid to the optimized medium increased biopolymer production from 47.2 to 66.1 g/L, the highest value reported in published literature with this strain. This simple, reproducible, and cheap fermentation process can considerably enhance the commercial applications of the poly-γ-glutamic acid synthesized by B. licheniformis ATCC 9945a.

A dynamic resazurin microassay allowing accurate quantification of cells and suitable for acid-forming bacteria.

A resazurin micro-assay was developed to quantify acidifying bacteria. The resorufin fluorescent signal was measured over time and the determined time to reach the max slope (TMS) was plotted against CFU (colony forming unit) counts. This dynamic assay enabled to quantify nine lactic acid bacteria and a Bacillus licheniformis strain despite the increasing acidity of the medium.

CRISPR-Cas9 mediated engineering of Bacillus licheniformis for industrial production of (2R,3S)-butanediol.

Bacillus lichenformis is an industrially promising generally recognized as safe (GRAS) strain that can be used for the production of a valuable chemical, 2,3-butanediol (BDO). Conventional gene deletion vectors and/or methods are time-consuming and have poor efficiency. Therefore, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 mediated homologous recombination was used to engineer a newly isolated and UV-mutagenized B. licheniformis 4071-15 strain. With the help of a CRISPR-Cas9 system, this one-step process could be used for the deletion of ldh gene within 4 days with high-efficiency exceeding 60%. In addition, the sequential deletion of target genes for engineering studies was evaluated, and it was confirmed that a triple mutant strain (ldh, dgp, and acoR) could be obtained by repeated one-step cycles. Furthermore, a practical metabolic engineering study was carried out using a CRISPR-Cas9 system for the stereospecific production of (2R,3S)-BDO. The predicted (2R,3R)-butanediol dehydrogenase encoded by the gdh gene was selected as a target for the production of (2R,3S)-BDO, and the mutant was successfully obtained. The results show that the stereospecific production of (2R,3S)-BDO was possible with the gdh deletion mutant, while the 4071-15 host strain still generated 26% of (2R,3R)-BDO. It was also shown that the 4071-15 Δgdh mutant could produce 115 g/L of (2R,3S)-BDO in 64 hr by two-stage fed-batch fermentation. This study has shown the efficient development of a (2R,3S)-BDO producing B. licheniformis strain based on CRISPR-Cas9 and fermentation technologies.

Antimicrobial activity as a potential factor influencing the predominance of Bacillus subtilis within the constitutive microflora of a whey reverse osmosis membrane biofilm.

Current cleaning and sanitation protocols may not be adequately effective in cleaning separation membranes and can result in the formation of resilient multispecies biofilms. The matured biofilms may result in a bacterial predominance with resilient strains on membranes with a prolonged use. In our previous study, we isolated organisms such as Bacillus subtilis, Bacillus licheniformis, Exiguobacterium aurantiacum, and Acinetobacter radioresistens from an 18-mo-old reverse osmosis membrane. The competitive exclusion studies revealed the predominance of B. subtilis within the membrane biofilm microflora. This study investigated the antimicrobial activity of the B. subtilis isolate as a potential cause of its predominance. The culture isolate was propagated in tryptic soy broth at 37°C, and microfiltered to prepare cell-free extracts (CFE) at 8-, 10-, 12-, 14-, 16-, and 18-h intervals. The CFE were freeze-dried and suspended in minimum quantities of HPLC-grade water to prepare concentrated solutions. The antimicrobial activities of CFE were tested using the agar-well assay against the biofilm constitutive microflora. The experiments were conducted in triplicates and means were compared for significant differences using a general linear mixed model procedure. The results indicated the highest antimicrobial activity of 12-h CFE of B. subtilis against other constitutive microflora such as Exiguobacterium sp., E. auranticum, and A. radioresistens, with average inhibition zone sizes of 16.5 ± 0.00, 16.25 ± 0.66, and 20.6 ± 0.00 mm, respectively. Upon treatment with proteinase K, the CFE completely lost its antimicrobial activity, establishing it to be a proteinaceous compound. The AA profiling revealed the total crude protein in CFE to be 51% (wt/wt), with its major constituent as glutamic acid (11.30% wt/wt). The freeze-dried CFE was thermally stable on exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min) and over a pH range of 3.0 to 6.3. The study helped us understand the role of the antimicrobial compound produced by B. subtilis as a potential cause of its predominance within the biofilm constitutive microflora.

Construction of a novel sugar alcohol-inducible expression system in Bacillus licheniformis.

Bacillus licheniformis is an important industrial microorganism that can utilize a wide range of biomass. However, the lack of expression elements in B. licheniformis, especially regulated promoters, significantly restricts its applications. In this study, two promoters involved in the sugar alcohol uptake pathway, PmtlA and PmtlR, were characterized and developed as regulated promoters for expression. The results showed that mannitol, mannose, sorbitol, sorbose, and arabinose can act as inducers to activate expression from PmtlA at different levels. The induction by sorbitol was the strongest, and the optimal induction conditions were 15 g/L sorbitol during mid-logarithmic growth at 28 °C. In this work, the palindrome-like sequence 'TTGTCA-cacggctcc-TGCCAA' in PmtlA was identified as the binding site of the MtlR protein. This study helps to enrich the known inducible expression systems in B. licheniformis.

Two genes involved in clindamycin resistance of Bacillus licheniformis and Bacillus paralicheniformis identified by comparative genomic analysis.

We evaluated the minimum inhibitory concentrations of clindamycin and erythromycin toward 98 Bacillus licheniformis strains isolated from several types of fermented soybean foods manufactured in several districts of Korea. First, based on recent taxonomic standards for bacteria, the 98 strains were separated into 74 B. licheniformis strains and 24 B. paralicheniformis strains. Both species exhibited profiles of erythromycin resistance as an acquired characteristic. B. licheniformis strains exhibited acquired clindamycin resistance, while B. paralicheniformis strains showed unimodal clindamycin resistance, indicating an intrinsic characteristic. Comparative genomic analysis of five strains showing three different patterns of clindamycin and erythromycin resistance identified 23S rRNA (adenine 2058-N6)-dimethyltransferase gene ermC and spermidine acetyltransferase gene speG as candidates potentially involved in clindamycin resistance. Functional analysis of these genes using B. subtilis as a host showed that ermC contributes to cross-resistance to clindamycin and erythromycin, and speG confers resistance to clindamycin. ermC is located in the chromosomes of strains showing clindamycin and erythromycin resistance and no transposable element was identified in its flanking regions. The acquisition of ermC might be attributable to a homologous recombination. speG was identified in not only the five genome-analyzed strains but also eight strains randomly selected from the 98 test strains, and deletions in the structural gene or putative promoter region caused clindamycin sensitivity, which supports the finding that the clindamycin resistance of Bacillus species is an intrinsic property.

Suboptimal Bacillus licheniformis and Bacillus weihenstephanensis Spore Incubation Conditions Increase Heterogeneity of Spore Outgrowth Time.

Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges of 4.7 to 7.4 and temperatures of 10°C to 37°C for B. weihenstephanensis and 18°C to 59°C for B. licheniformis Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, a lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage. The heat treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C for 12 min or at 95°C for 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite the fact that they both led to a 10-fold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth 1.2-fold and lowered the proportion of spores able to germinate and outgrow 1.4-fold. Interestingly, we showed by proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 than in those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH.IMPORTANCE Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sublethal heat treatment. Using flow cytometry, we were able to follow at a single-cell level the changes in the physiological states of heat-stressed spores of Bacillus spp. and to discriminate between dormant spores, germinated spores, and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sublethal heat treatment, a common situation in food processing and a concern for food preservation and safety.

Establishing a Fed-Batch Process for Protease Expression with Bacillus licheniformis in Polymer-Based Controlled-Release Microtiter Plates.

Introducing fed-batch mode in early stages of development projects is crucial for establishing comparable conditions to industrial fed-batch fermentation processes. Therefore, cost efficient and easy to use small-scale fed-batch systems that can be integrated into existing laboratory equipment and workflows are required. Recently, a novel polymer-based controlled-release fed-batch microtiter plate is described. In this work, the polymer-based controlled-release fed-batch microtiter plate is used to investigate fed-batch cultivations of a protease producing Bacillus licheniformis culture. Therefore, the oxygen transfer rate (OTR) is online-monitored within each well of the polymer-based controlled-release fed-batch microtiter plate using a µRAMOS device. Cultivations in five individual polymer-based controlled-release fed-batch microtiter plates of two production lots show good reproducibility with a mean coefficient of variation of 9.2%. Decreasing initial biomass concentrations prolongs batch phase while simultaneously postponing the fed-batch phase. The initial liquid filling volume affects the volumetric release rate, which is directly translated in different OTR levels of the fed-batch phase. An increasing initial osmotic pressure within the mineral medium decreases both glucose release and protease yield. With the volumetric glucose release rate as scale-up criterion, microtiter plate- and shake flask-based fed-batch cultivations are highly comparable. On basis of the small-scale fed-batch cultivations, a mechanistic model is established and validated. Model-based simulations coincide well with the experimentally acquired data.

Optimized production of poly (γ-glutamic acid) (γ-PGA) using Bacillus licheniformis and its application as cryoprotectant for probiotics.

Bacteria produce poly (γ-glutamic acid) (γ-PGA), a polymer of l- or d-glutamic acid, as a defense response and have gained importance due to their applications in food and pharmaceutical industry. In the present investigation, production of γ-PGA using cost-effective carbon substrate, characterization of the produced polymer, and its application as cryoprotectant for selected freeze-dried probiotic bacteria were investigated. Central composite rotatable design of response surface methodology was used to study the main and the interactive effects of medium components: rice bran and casein peptone concentration. Rice bran at 35% (w/v) and casein peptone at 7.5% (w/v) were found to be optimal at an initial pH of 7.5, and incubation temperature of 37°C for 48 H produced 8.2 g/L γ-PGA on dry weight basis. The thermal properties such as melting temperature, heat of fusion, and thermal stability were also studied. Ten percent (w/v) of γ-PGA with 10 percent of sodium alginate (w/v) protected viability of Bifidiobacterium bifidum NCDC 235 and B. adolescentis NCDC 236 during freeze drying at -80 ËšC for 48 H. The γ-PGA synthesized by the reported bacterium with GRAS status is suitable for food and biomedical applications.

Production and Application of Biosurfactant Produced by Bacillus Licheniformis Ali5 in Enhanced Oil Recovery and Motor Oil Removal from Contaminated Sand.

The present study describes the production of biosurfactant from isolate B. licheniformis Ali5. Seven different, previously-reported minimal media were screened for biosurfactant production, and two selected media were further optimized for carbon source. Further, various fermentation conditions such as (pH 2-12, temperature 20-50 °C, agitation speed 100-300 rpm, NaCl (0-30 g·L-1) were investigated. The partially purified biosurfactant was characterized by Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) and found a lipopeptide mixture, similar to lichenysin-A. Biosurfactant reduced surface tension from 72.0 to 26.21 ± 0.3 and interfacial tension by 0.26 ± 0.1 mN.m-1 respectively, biosurfactant yield under optimized conditions was 1 g·L-1, with critical micelle concentration (CMC) of 21 mg·L-1 with high emulsification activity of (E24) 66.4 ± 1.4% against crude oil. Biosurfactant was found to be stable over extreme conditions. It also altered the wettability of hydrophobic surface by changing the contact angle from 49.76° to 16.97°. Biosurfactant efficiently removed (70-79%) motor oil from sand, with an efficiency of more than 2 fold as compared without biosurfactant (36-38%). It gave 32% additional oil recovery over residual oil saturation upon application to a sand-packed column. These results are indicative of potential application of biosurfactant in wettability alteration and ex-situ microbial enhanced oil recovery.

Algicidal characterization and mechanism of Bacillus licheniformis Sp34 against Microcystis aeruginosa in Dianchi Lake.

Microcystis aeruginosa blooms are a worldwide serious environmental problem and bloom control with bacteria is promising. In this study, a Bacillus licheniformis strain Sp34 with potent algicidal and inhibitory effects on the microcystins synthesis against fast-growing M. aeruginosa was isolated from Dianchi Lake. Sp34 killed the bloom-causing algal strain M. aeruginosa DCM4 of Dianchi Lake with an initial Chlorophyll-a concentration of 2.0 mg/L at a cell density of no less than 1.35 × 105 CFU/ml. It can also efficiently kill some other harmful algal species, such as M. wesenbergii and Phormidium sp. The algicidal activity of Sp34 relied on the release of algicidal substances, which had good heat (-20°C to 121°C) and acid-base (pH 3-11) resistance. In addition, the high algicidal activity depended on the good growth of algae indicated by the significantly positive correlations between algal growth and algicidal ratio (p < .001). The algicidal effect of Sp34 involved causing oxidative stress, lipid peroxidation, and morphological injury of algal cells, along with DNA damage and dysfunction of DNA-repair function, weakening the photosynthesis system, and inhibiting microcystin synthesis. In general, Sp34 can kill fast-growing M. aeruginosa and inhibit algal microcystin synthesis efficiently, so, it is a promising biocontrol agent to mitigate cyanobacterial blooms.

Combined dissolved oxygen tension and online viscosity measurements in shake flask cultivations via infrared fluorescent oxygen-sensitive nanoparticles.

Oxygen supply is one of the most critical process parameters in aerobic cultivations. To assure sufficient oxygen supply, shake flasks are usually used in combination with orbital shaking machines. In this study, a measurement technique for the dissolved oxygen tension (DOT) in shake flask cultures with viscosity changes is presented. The movement of the shaker table is monitored by means of a Hall effect sensor. For DOT measurements, infrared fluorescent oxygen-sensitive nanoparticles are added to the culture broth. The position of the rotating bulk liquid needs to be determined to assure measurements inside the liquid. The leading edge of the bulk liquid is detected based on the fluorescence signal intensity of the oxygen-sensitive nanoparticles. Furthermore, online information about the viscosity of the culture broth is acquired due to the detection of the position of the leading edge of the bulk liquid relative to the direction of the centrifugal force, as described by Sieben et al. (2019. Sci. Rep., 9, 8335). The DOT measurement is combined with a respiration activity monitoring system which allows for the determination of the oxygen transfer rate (OTR) in eight parallel shake flasks. Based on DOT and OTR, the volumetric oxygen transfer coefficient (kL a) is calculated during cultivation. The new system was successfully applied in cultivations of Escherichia coli, Bacillus licheniformis, and Xanthomonas campestris.

Knockout of pgdS and ggt gene changes poly-γ-glutamic acid production in Bacillus licheniformis RK14-46.

Poly-gamma-glutamic acid (γ-PGA) is a water-soluble, nontoxic biocompatible polymer, which is extensively used in medicines, foodstuffs, cosmetics, and in water treatment. We previously isolated a novel γ-PGA producing strain Bacillus licheniformis RK14 from soil and developed a hyper-producing mutant strain RK14-46 by an ethyl methanesulfonate (EMS) treatment. In this study, endo-type (pgdS) and exo-type γ-PGA hydrolases (ggt) were disrupted by integrating plasmids into the genomic DNA of B. licheniformis RK14-46 strain. Unexpectedly, we observed strong inhibition of γ-PGA production following deletion of the pgdS gene, suggesting that pgdS is essential for γ-PGA biosynthesis in strain RK14-46, and in its parent strain RK14. In contrast, γ-PGA production increased by the deletion of the ggt gene and reached 39 g/L in the presence of 90 g/L glucose and elevated oxygen supply. Furthermore, γ-PGA from the ggt-disrupted mutant (Δggt) maintained a larger molecular mass throughout the culture period, whereas that from the original RK14-46 strain had degraded after glucose consumption. γ-PGA-containing culture supernatants from Δggt strain showed greater flocculation efficiency in sewage sludge than supernatants from the RK14-46 strain, reflecting greater production of γ-PGA with larger molecular mass by the Δggt strain. This is the first report concerning the deletion of pgdS and ggt genes in B. licheniformis strain and the properties of γ-PGA obtained from the mutant strain.

Shake flask methodology for assessing the influence of the maximum oxygen transfer capacity on 2,3-butanediol production.

BACKGROUND: Production of 2,3-butanediol from renewable resources is a promising measure to decrease the consumption of fossil resources in the chemical industry. One of the most influential parameters on biotechnological 2,3-butanediol production is the oxygen availability during the cultivation. As 2,3-butanediol is produced under microaerobic process conditions, a well-controlled oxygen supply is the key parameter to control biomass formation and 2,3-butanediol production. As biomass is on the one hand not the final product, but on the other hand the essential biocatalyst, the optimal compromise between biomass formation and 2,3-butanediol production has to be defined. RESULTS: A shake flask methodology is presented to evaluate the effects of oxygen availability on 2,3-butanediol production with Bacillus licheniformis DSM 8785 by variation of the filling volume. A defined two-stage cultivation strategy was developed to investigate the metabolic response to different defined maximum oxygen transfer capacities at equal initial growth conditions. The respiratory quotient was measured online to determine the point of glucose depletion, as 2,3-butanediol is consumed afterwards. Based on this strategy, comparable results to stirred tank reactors were achieved. The highest space-time yield (1.3 g/L/h) and a 2,3-butanediol concentration of 68 g/L combined with low acetoin concentrations and avoided glycerol formation were achieved at a maximum oxygen transfer capacity of 13 mmol/L/h. The highest overall 2,3-butanediol concentration of 78 g/L was observed at a maximum oxygen transfer capacity of 4 mmol/L/h. CONCLUSIONS: The presented shake flask approach reduces the experimental effort and costs providing a fast and reliable methodology to investigate the effects of oxygen availability. This can be applied especially on product and by-product formation under microaerobic conditions. Utilization of the maximum oxygen transfer capacity as measure for the oxygen availability allows for an easy adaption to other bioreactor setups and scales.

Effective biodegradation of chicken feather waste by co-cultivation of keratinase producing strains.

BACKGROUND: Chicken feather, a byproduct of poultry-processing industries, are considered a potential high-quality protein supplement owing to their crude protein content of more than 85%. Nonetheless, chicken feathers have been classified as waste because of the lack of effective recycling methods. In our previous studies, Bacillus licheniformis BBE11-1 and Stenotrophomonas maltophilia BBE11-1 have been shown to have feather-degrading capabilities in the qualitative phase. To efficiently recycle chicken feather waste, in this study, we investigated the characteristics of feather degradation by B. licheniformis BBE11-1 and S. maltophilia BBE11-1. In addition, in an analysis of the respective advantages of the two degradation systems, cocultivation was found to improve the efficiency of chicken feather waste degradation. RESULTS: B. licheniformis BBE11-1 and S. maltophilia BBE11-1 were used to degrade 50 g/L chicken feather waste in batches, and the degradation rates were 35.4% and 22.8% in 96 h, respectively. The degradation rate of the coculture system reached 55.2% because of higher keratinase and protease activities. Furthermore, cocultivation was conducted in a 3 L fermenter by integrating dissolved oxygen control and a two-stage temperature control strategy. Thus, the degradation rate was greatly increased to 81.8%, and the conversion rate was 70.0% in 48 h. The hydrolysates exhibited antioxidant activity and contained large quantities of amino acids (895.89 mg/L) and soluble peptides. CONCLUSIONS: Cocultivation of B. licheniformis BBE11-1 and S. maltophilia BBE11-1 can efficiently degrade 50 g/L chicken feather waste and produce large amounts of amino acids and antioxidant substances at a conversion rate of 70.0%.

Sporulating behavior of Bacillus licheniformis strains influences their population dynamics during raw milk holding.

To understand the role of strain variability, population dynamics of 2 strains of Bacillus licheniformis, ATCC 6634 and ATCC 14580, were modeled as a function of temperature (4.0-12.0°C) and duration (0-72 h) using regression analysis. Based on the initial spiking of vegetative cells (approximately 4.0 log cfu/mL) and spores (approximately 2.0 log cfu/mL), regression equations, elucidating B. licheniformis growth behavior during raw milk holding at low temperature, were obtained. Contour plots were developed to determine the time-temperature combinations, keeping the population changes to less than 1.0 log. In vegetative cell spiking study of B. licheniformis ATCC 6634 (S1), cell population changes remained below 1.0 log up to 72 h at 8°C. For B. licheniformis ATCC 14580 (S2), 1.0 log shift was not observed only after 80 h at 8°C, indicating higher multiplication potential of S1 as compared with S2. As S2 was a readily sporulating strain, the vegetative spiking study showed spore formation at different storage temperatures. Evidence of some parallel germination was observed for this strain at 8°C or higher, when raw milk samples were spiked with spores. The experimental values obtained for sporeformers and spore counts were validated with contour plot-generated values. Overall, for raw milk samples predominated by the low sporulating strain, the contour plots suggested holding at 8°C or below for up to 72 h. In the case of the readily sporulating strain (S2), raw milk could be held at 8°C for 80 h, where little or no sporulation is observed. Sporulation behavior, germination and multiplication ability, strain variability, and temperature and duration of holding raw milk influenced the population dynamics of Bacillus species. However, in the presence of equivalent numbers of both types of sporulating strains in raw milk, despite strain variability, holding milk at 8°C for not more than 72 h would keep any cell population changes below 1.0 log. In addition, under these storage conditions, the population would remain as vegetative cells that are likely to be inactivated by pasteurization. The contour plots, so generated, would help predict the population shifts and define optimum holding conditions for raw milk before further processing.

The Influence of Temperature and Nitrogen Source on Cellulolytic Potential of Microbiota Isolated from Natural Environment.

Bacteria from the genus Bacillus are a rich source of commercial enzymes, including amylases, proteases, cellulases, glucose isomerase, and pullulanase. Cellulases account for 15% of the global market of industrial enzymes; thus, new microorganisms producing cellulases in a higher concentration and new ingredients, which can enhance the level of enzyme synthesis, are still needed. Many of cellulose-degrading microorganisms have been isolated so far and characterized in various regions of the world. In this study, we were looking for the bacteria isolated from the natural environment with the high cellulolytic potential, which could be used as components of a biopreparation to accelerate decomposition of postharvest leftovers in agriculture. The 214 bacterial strains were isolated from environmental samples rich in cellulose and their ability to synthesize cellulases were examined using the diffusion method. Six strains, which have the highest diameter of clearing zone both for biomass and supernatant, were selected for identification. Optimization of biosynthesis of the cellulose-degrading enzymes indicated that optimal temperature of this process fluctuated in the range of 21-42°C (depending on the strain and carbon source). The highest cellulolytic activity was observed for the isolates designed as 4/7 (identified as Bacillus subtilis) and 4/18 (identified as Bacillus licheniformis) in a temperature of 32°C. With the use of a desirability function methodology, the optimal medium composition to achieve a simple, cost-efficient process of cellulases production was developed for both strains. These experiments show that microorganisms isolated from natural environmental samples have unique properties and potential for commercial applications (e.g. for biopreparations production).Bacteria from the genus Bacillus are a rich source of commercial enzymes, including amylases, proteases, cellulases, glucose isomerase, and pullulanase. Cellulases account for 15% of the global market of industrial enzymes; thus, new microorganisms producing cellulases in a higher concentration and new ingredients, which can enhance the level of enzyme synthesis, are still needed. Many of cellulose-degrading microorganisms have been isolated so far and characterized in various regions of the world. In this study, we were looking for the bacteria isolated from the natural environment with the high cellulolytic potential, which could be used as components of a biopreparation to accelerate decomposition of postharvest leftovers in agriculture. The 214 bacterial strains were isolated from environmental samples rich in cellulose and their ability to synthesize cellulases were examined using the diffusion method. Six strains, which have the highest diameter of clearing zone both for biomass and supernatant, were selected for identification. Optimization of biosynthesis of the cellulose-degrading enzymes indicated that optimal temperature of this process fluctuated in the range of 21­42°C (depending on the strain and carbon source). The highest cellulolytic activity was observed for the isolates designed as 4/7 (identified as Bacillus subtilis) and 4/18 (identified as Bacillus licheniformis) in a temperature of 32°C. With the use of a desirability function methodology, the optimal medium composition to achieve a simple, cost-efficient process of cellulases production was developed for both strains. These experiments show that microorganisms isolated from natural environmental samples have unique properties and potential for commercial applications (e.g. for biopreparations production).