Efficient biodegradation of organic matter using a thermophilic bacterium and development of a cost-effective culture medium for industrial use.

Microorganisms with efficient organic matter degradation ability are essential for organic waste treatment. In this study, a thermophilic bacterium, Bacillus thermoliquefaciens, was identified to have excellent cellulase, amylase, and protease activity, and provided efficient degradation of food waste. This is the first report on the organic matter degradation potential of B. thermoliquefaciens. Using a "one-variable-at-a-time" approach and response surface methodology, the optimal culture conditions for B. thermoliquefaciens were determined to be a 5% inoculation level, 50 °C culture temperature, 25 mL filling volumes in 250 mL flasks, and 180 rpm shaking for 24 h. The optimized medium was formulated as 1 g Na2HPO4, 1 g KH2PO4, 0.05 g MgSO4, 3 g NaCl, 0.05 g CaCl2, 11.44 g wheat bran powder, 4.92 g soybean meal, and 1 L distilled water at pH 7.12. The maximum biomass attained was 1.57 ± 0.153 × 109 CFU/mL. The cost of this medium was 4.18 times less than that before optimization. This promising result lays a foundation for future industrial application of this bacterium to the degradation of organic waste.

Low-cost cultivation and sporulation of alkaliphilic Bacillus sp. strain AK13 for self-healing concrete.

The alkaliphilic, calcium carbonate precipitating Bacillus sp. strain AK13 can be utilized in concrete for self-repairing. A statistical experimental design was used to develop an economical medium for its mass cultivation and sporulation. Two types of screening experiment were first conducted to identify substrates that promote the growth of the AK13 strain: the first followed a one-factor-at-a-time factorial design and the second a two-level full factorial design. Based on these screening experiments, barley malt powder and mixed grain powder were identified as the substrates that most effectively promoted the growth of the AK13 strain from a range of 21 agricultural products and by-products. A quadratic statistical model was then constructed using a central composite design and the concentration of the two substrates was optimized. The estimated growth and sporulation of Bacillus sp. strain AK13 in the proposed medium were 3.08 ± 0.38 × 108 and 1.25 ± 0.12 × 108 CFU/ml, respectively, which meant that the proposed low-cost medium was approximately 45 times more effective than the commercial medium in terms of the number of cultivatable bacteria per unit price. The spores were then powdered via a spray-drying process to produce a spore powder with a spore count of 2.0 ± 0.7 × 109 CFU/g. The AK13 spore powder was mixed with cement paste, yeast extract, calcium lactate, and water. The yeast extract and calcium lactate generated the highest CFU/ml for AK13 at a 0.4:0.4 ratio compared to 0.4:0.25 (the original ratio of the B4 medium) and 0.4:0.8. Twenty-eight days after the spores were mixed into the mortar, the number of vegetative cells and spores of the AK13 strain had reached 106 CFU/g within the mortar. Cracks in the mortar under 0.29 mm were healed in 14 days. Calcium carbonate precipitation was observed on the crack surface. The mortar containing the spore powder was thus concluded to be effective in terms of healing micro-cracks.

Stochastic modeling of variability in survival behavior of Bacillus simplex spore population during isothermal inactivation at the single cell level using a Monte Carlo simulation.

The control of bacterial reduction is important to maintain food safety during thermal processing. The goal of this study was to illustrate and describe variability in bacterial population behavior during thermal processing as a probability distribution based on individual cell heterogeneity regarding heat resistance. Toward this end, we performed a Monte Carlo simulation via computer, and compared and validated the simulated estimations with observed values. Weibullian fitted parameters were estimated from the kinetic survival data of Bacillus simplex during thermal treatment at 94 °C. The variability in reductions of bacterial sporular populations was illustrated using Monte Carlo simulation based on the Weibull distribution of the parameters. In particular, variabilities in viable spore counts and survival probability of the B. simplex spore population were simulated in various replicates. We also experimentally determined the changes in survival probability and distributions of survival spore counts; notably, these were successfully predicted by the Monte Carlo simulation based on the kinetic parameters. The kinetic parameter-based Monte Carlo simulation could thus successfully illustrate bacterial population behavior variability during thermal processing as a probability distribution. The simulation approach may contribute to improving food quality through risk-based processing designs and enhance risk assessment model accuracy.

Screening of novel bacteria for the 2,3-butanediol production.

Biotechnologically produced 2,3-butanediol (2,3-BDO) is a potential starting material for industrial bulk chemicals such as butadiene or methyl ethyl ketone which are currently produced from fossil feedstocks. So far, the highest 2,3-BDO concentrations have been obtained with risk group 2 microorganisms. In this study, three risk group 1 microorganisms are presented that are so far unknown for an efficient production of 2,3-BDO. The strains Bacillus atrophaeus NRS-213, Bacillus mojavensis B-14698, and Bacillus vallismortis B-14891 were evaluated regarding their ability to produce high 2,3-BDO concentrations with a broad range of different carbon sources. A maximum 2,3-BDO concentration of 60.4 g/L was reached with the strain B. vallismortis B-14891 with an initial glucose concentration of 200 g/L within 55 h in a batch cultivation. Besides glucose, B. vallismortis B-14891 converts 14 different substrates that can be obtained from residual biomass sources to 2,3-BDO. Therefore B. vallismortis B-14891 is a promising candidate for the large-scale production of 2,3-BDO with low-cost substrates.

A cost effective cultivation medium for biocalcification of Bacillus pasteurii KCTC 3558 and its effect on cement cubes properties.

Application of carbonate precipitation induced by Bacillus pasteurii for improving some properties of cement has been reported. However, it is not yet successful in commercial scale due to the high cost of cultivation medium. This is the first report on the application of effluent from chicken manure bio-gas plant, a high protein content agricultural waste, as an alternative growth medium for carbonate precipitation by B. pasteurii KCTC3558. Urease activity of B. pasteurii KCTC3558 cultured in chicken manure effluent medium and other three standard media were examined using phenate method. The highest urease production was achieved in chicken manure effluent medium (16.756Umg(-1) protein). Cost per liter of chicken manure effluent medium is up to 88.2% lower than other standard media. The most effective cultivation media was selected for carbonate precipitation study in cement cubes. Water absorption, voids, apparent density and compressive strength of cement cubes were measured according to the ASTM standard. The correlation between the increasing density and compressive strength of bacterial added cement cube was evident. The density of bacterial cement cube is 5.1% higher than control while the compressive strength of cement mixed with bacterial cells in chicken manure effluent medium increases up to 30.2% compared with control. SEM and XRD analysis also found the crystalline phase of calcium carbonate within bacterial cement which confirmed that the increasing density and compressive strength were resulted from bacterial carbonate precipitation. This study indicated that the effluent from chicken manure bio-gas plant could be used as an alternative cost effective culture medium for cultivation and biocalcification of B. pasteurii KCTC3558 in cement.

Survival of Bacillus spp. SUBB01 at high temperatures and a preliminary assessment of its ability to protect heat-stressed Escherichia coli cells.

BACKGROUND: The bacterial stressed state upon temperature raise has widely been observed especially in Escherichia coli cells. The current study extended such physiological investigation on Bacillus spp. SUBB01 under aeration at 100 rpm on different culture media along with the high temperature exposure at 48, 50, 52, 53 and 54 °C. Bacterial growth was determined through the enumeration of the viable and culturable cells; i.e., cells capable of producing the colony forming units on Luria-Bertani and nutrient agar plates up to 24 h. Microscopic experiments were conducted to scrutinize the successive physiological changes. Suppression of bacterial growth due to the elevated heat was further confirmed by the observation of non-viability through spot tests. RESULTS: As expected, a quick drop in both cell turbidity and colony forming units (~10(4)) along with spores were observed after 12-24 h of incubation period, when cells were grown at 54 °C in both Luria-Bertani and nutrient broth and agar. The critical temperature (the temperature above which it is no longer possible to survive) of Bacillus spp. SUBB01 was estimated to be 53 °C. Furthermore, a positive impact was observed on the inhibited E. coli SUBE01 growth at 45 and 47 °C, upon the supplementation of the extracellular fractions of Bacillus species into the growing culture. CONCLUSIONS: Overall the present analysis revealed the conversion of the culturable cells into the viable and nonculturable (VBNC) state as a result of heat shock response in Bacillus spp. SUBB01 and the cellular adaptation at extremely high temperature.

In vitro assessment of marine Bacillus for use as livestock probiotics.

Six antimicrobial-producing seaweed-derived Bacillus strains were evaluated in vitro as animal probiotics, in comparison to two Bacillus from an EU-authorized animal probiotic product. Antimicrobial activity was demonstrated on solid media against porcine Salmonella and E. coli. The marine isolates were most active against the latter, had better activity than the commercial probiotics and Bacillus pumilus WIT 588 also reduced E. coli counts in broth. All of the marine Bacillus tolerated physiological concentrations of bile, with some as tolerant as one of the probiotics. Spore counts for all isolates remained almost constant during incubation in simulated gastric and ileum juices. All of the marine Bacillus grew anaerobically and the spores of all except one isolate germinated under anaerobic conditions. All were sensitive to a panel of antibiotics and none harbored Bacillus enterotoxin genes but all, except B. pumilus WIT 588, showed some degree of ß-hemolysis. However, trypan blue dye exclusion and xCELLigence assays demonstrated a lack of toxicity in comparison to two pathogens; in fact, the commercial probiotics appeared more cytotoxic than the majority of the marine Bacillus. Overall, some of the marine-derived Bacillus, in particular B. pumilus WIT 588, demonstrate potential for use as livestock probiotics.

Glycerol-based sterilization bioindicator system from Bacillus atrophaeus: development, performance evaluation, and cost analysis.

The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D (160 °C) value of 6.6 ± 0.1 min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3 ± 1.2 × 10(8) CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48 h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24 h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8 % (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.

Bioflocculant production from Solibacillus silvestris W01 and its application in cost-effective harvest of marine microalga Nannochloropsis oceanica by flocculation.

Microalgae are widely studied for biofuel production, however, current technologies to harvest microalgae for this purpose are not well developed. In this work, a bacterial strain W01 was isolated from activated sludge and identified as Solibacillus silvestris. Bioflocculant in the culture broth of W01 showed 90% flocculating efficiency on marine microalga Nannochloropsis oceanica, and no metal ion was required for the flocculation process. Chemical analysis of the purified bioflocculant indicated that it is a proteoglycan composed of 75.1% carbohydrate and 24.9% protein (w/w). The bioflocculant exhibits no effect on the growth of microalgal cells and can be reused to for economical harvesting of N. oceanica. This is the first report that strain of S. silvestris can produce bioflocculant for microalgae harvest. The novel bioflocculant produced by W01 has the potential to harvest marine microalgae for cost-effective production of microalgal bioproducts.

Development of a low-cost sterilization biological indicator using Bacillus atrophaeus by solid-state fermentation.

The production of biological indicators involving bacterial sporulation and multi-step downstream processes has been described. The goal of the present work was to use fermented material as the final product in a biological indicator, thereby reducing processing steps and costs. The performance of three different inexpensive supports (vermiculite, sand, and sugarcane bagasse) was assessed by determining Bacillus atrophaeus sporulation during solid-state fermentation and by assessing the direct use of the fermentation products in the subsequent steps of the process. All three supports allowed spore production of between 10(7) and 10(9) CFU g(-1). Sand proved to be the best inert support enabling the direct use of the fermented product due to its easy homogenization, filling properties, and compatibility with recovery medium. Bacterial adhesion to the sand surface was supported by biofilm formation. The resistance to sterilization of the dried fermentation product was evaluated. For dry-heat resistance (160°C), the D value was 6.6 min, and for ethylene oxide resistance (650 mg/L), the D value was 6.5 min. The cost reduction of this process was at least 48%. No previous studies have been published on the application of sand as a support in solid-state fermentation for the production of biological indicators.

Coffee husk waste for fermentation production of mosquitocidal bacteria.

Coffee husk waste (CHW) discarded as bio-organic waste, from coffee industries, is rich in carbohydrates. The current study emphasizes the management of solid waste from agro-industrial residues for the production of biopesticides (Bacillus sphaericus, and B. thuringiensis subsp. israelensis), to control disease transmitting mosquito vectors. An experimental culture medium was prepared by extracting the filtrates from coffee husk. A conventional culture medium (NYSM) also was prepared. The studies revealed that the quantity of mosquitocidal toxins produced from CHW is at par with NYSM. The bacteria produced in these media, were bioassayed against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti) and it was found that the toxic effect was statistically comparable. Cost-effective analysis have revealed that, production of biopesticides from CHW is highly economical. Therefore, the utilization of CHW provides dual benefits of effective utilization of environmental waste and efficient production of mosquitocidal toxins.

Is the availability of different nutrients a critical factor for the impact of bacteria on subterraneous carbon budgets?

Bacteria thriving in underground systems, such as karsts, adapt to use a variety of nutrients. Most of these nutrients derive from superficial processes. This study shows that bacteria are able to differentially induce carbonate precipitation or dissolution depending on the availability of nutrients for growth. Different bacterial strains isolated from caves, representing the most common components of these microbial communities, were cultured with different carbon and nitrogen sources (e.g., acetate, glucose, peptone, humic acids) and induced changes in pH were measured during growth. Carbonate can either precipitate or dissolve during bacterial growth. The induction of carbonate precipitates or their dissolution as a function of consumption of specific carbon sources revealed the existence of an active nutrient cycling process in karsts and links nutrients and environmental conditions to the existence of a highly significant carbon sink in subterraneous environments.

Egg yolk enhances early sporulation and toxicity of Bacillus sphaericus H5a5b for small-scale production of a mosquito control agent.

Bacillus sphaericus has been widely used in mosquito control programs, but the production of this bacterium is a little tricky as it does not utilize carbohydrates and requires proteinaceous substrates, which are expensive. In this study, we developed a cost-effective medium that resulted in a lower cost and shorter fermentation time. The locally available raw material, egg yolk was used and the level of sporulation, toxicity and biomass were compared with the conventional medium. Use of the egg yolk culture medium significantly shortened fermentation time to 15 h and yielded high activity, equivalent to that of conventional medium against 3rd instar Culex quinquefasciatus. Conventional NYSM medium required 21 h to attain the maximum activity and biomass. Hence, the egg yolk-based culture medium appears to be suitable and economical for the small-scale production of B. sphaericus.

Selection and optimization of Bacillus atrophaeus inoculum medium and its effect on spore yield and thermal resistance.

Bacillus atrophaeus's spores are used as biological indicators to monitor sterilization processes and as a Bacillus anthracis surrogate in the development and validation of biosafety methods. The regular use of biological indicators to evaluate the efficiency of sterilization processes is a legal requirement for health services. However, its high cost hinders its widespread use. Aiming at developing a cost-effective inoculum medium, soybean molasses and nutrient-supplemented vinasse were evaluated for their effectiveness in solid-state fermentation (SSF). In biomass production, the results demonstrated that all tested compositions favor growth by providing the nutritional demands of the microorganism. Optimum casein peptone and soybean molasses concentration (1.0%, 2.5%, or 4.0%) was determined by a 2((2-0)) factorial experimental design. The results have showed a positive influence of peptone on biomass production. In order to define peptone final concentration (4.0% or 6.0%), a 2(2) factorial experimental design was used. An optimized medium containing 4.0% soybean molasses and 4.0% casein peptone was similar in performance to a synthetic control medium (tryptone soy broth) in dry-heat thermal-resistant spore production by SSF. An experiment performed under optimum SSF conditions resulted in 1.9 x 10(10) CFU g(-1) dry matter with D (160 degrees C) = 5.2 +/- 0.2 min.

Assessment of the role of chemotaxis and biofilm formation as requirements for colonization of roots and seeds of soybean plants by Bacillus amyloliquefaciens BNM339.

This article correlates colonization with parameters, such as chemotaxis, biofilm formation, and bacterial growth, that are believed to be connected. We show here, by using two varieties of soybean plants that seeds axenically produced exudates, induced a chemotactic response in Bacillus amyloliquefaciens, whereas root exudates did not, even when the exudates, also collected under axenic conditions, were concentrated up to 200-fold. Root exudates did not support bacterial cell division, whereas seed exudates contain compounds that support active cell division and high cell biomass at stationary phase. Seed exudates of the two soybean varieties also induced biofilm formation. B. amyloliquefaciens colonized both seeds and roots, and plant variety significantly affected bacterial root colonization, whereas it did not affect seed colonization. Colonization of roots in B. amyloliquefaciens occurred despite the lack of chemotaxis and growth stimulation by root exudates. The data presented in this article suggest that soybean seed colonization, but not root colonization, by B. amyloliquefaciens is influenced by chemotaxis, growth, and biofilm formation and that this may be caused by qualitative changes of the composition of root exudates.

Assessment of heat resistance of bacterial spores from food product isolates by fluorescence monitoring of dipicolinic acid release.

This study is aimed at the development and application of a convenient and rapid optical assay to monitor the wet-heat resistance of bacterial endospores occurring in food samples. We tested the feasibility of measuring the release of the abundant spore component dipicolinic acid (DPA) as a probe for heat inactivation. Spores were isolated from the laboratory type strain Bacillus subtilis 168 and from two food product isolates, Bacillus subtilis A163 and Bacillus sporothermodurans IC4. Spores from the lab strain appeared much less heat resistant than those from the two food product isolates. The decimal reduction times (D values) for spores from strains 168, A163, and IC4 recovered on Trypticase soy agar were 1.4, 0.7, and 0.3 min at 105 degrees C, 120 degrees C, and 131 degrees C, respectively. The estimated Z values were 6.3 degrees C, 6.1 degrees C, and 9.7 degrees C, respectively. The extent of DPA release from the three spore crops was monitored as a function of incubation time and temperature. DPA concentrations were determined by measuring the emission at 545 nm of the fluorescent terbium-DPA complex in a microtiter plate fluorometer. We defined spore heat resistance as the critical DPA release temperature (Tc), the temperature at which half the DPA content has been released within a fixed incubation time. We found Tc values for spores from Bacillus strains 168, A163, and IC4 of 108 degrees C, 121 degrees C, and 131 degrees C, respectively. On the basis of these observations, we developed a quantitative model that describes the time and temperature dependence of the experimentally determined extent of DPA release and spore inactivation. The model predicts a DPA release rate profile for each inactivated spore. In addition, it uncovers remarkable differences in the values for the temperature dependence parameters for the rate of spore inactivation, DPA release duration, and DPA release delay.

An assessment of pasteurization treatment of water, media, and milk with respect to Bacillus spores.

This study evaluated the ability of spore-forming Bacillus spp. to resist milk pasteurization conditions from 72 to 150 degrees C. Spores from the avirulent surrogate Sterne strain of Bacillus anthracis, as well as a representative strain of a common milk contaminant that is also a pathogen, Bacillus cereus ATCC 9818, were heated at test temperatures for up to 90 min in dH2O, brain heart infusion broth, or skim milk. In skim milk, characteristic log reductions (log CFU per milliliter) for B. anthracis spores were 0.45 after 90 min at 72 degrees C, 0.39 after 90 min at 78 degrees C, 8.10 after 60 min at 100 degrees C, 7.74 after 2 min at 130 degrees C, and 7.43 after 0.5 min at 150 degrees C. Likewise, log reductions (log CFU per milliliter) for viable spores of B. cereus ATCC 9818 in skim milk were 0.39 after 90 min at 72 degrees C, 0.21 after 60 min at 78 degrees C, 7.62 after 60 min at 100 degrees C, 7.37 after 2 min at 130 degrees C, and 7.53 after 0.5 min at 150 degrees C. No significant differences (P < 0.05) in thermal resistance were observed for comparisons of spores heated in dH2O or brain heart infusion broth compared with results observed in skim milk for either strain tested. However, spores from both strains were highly resistant (P < 0.05) to the pasteurization temperatures tested. As such, pasteurization alone would not ensure complete inactivation of these spore-forming pathogens in dH2O, synthetic media, or skim milk.

Microbiologic quality-control study for the purpose of extending the use of transfer sets on the automix 3 + 3 and micromix automated total nutrient admixture compounding pumps.

BACKGROUND: A quality-control study was undertaken by the departments of pharmacy and microbiology at St. Paul's Hospital (Vancouver, British Columbia, Canada) to evaluate the microbiologic safety of total nutrition admixtures (TNA) compounded by automated compounding pumps when the use of disposable transfer sets was extended from 1 day to 2 days. This study also evaluates the potential annual cost savings of this extended use. METHODS: Transfer sets and unused part containers of ingredients were left to sit overnight on the automated compounders after daily TNA manufacturing before a TNA sample was compounded for culturing. These TNA samples were cultured using a biphasic system consisting of a tryptic soy broth component and an agar slide component. Positive results were subcultured and isolates were identified by standard methods. Forty samples were collected and evaluated. RESULTS: Four bags grew Bacillus species, and 1 bag grew coagulase-negative staphylococci. The potential annual cost savings of this extended use was estimated to be approximately 35,000 Canadian dollars. CONCLUSIONS: The extended use of the disposable transfer sets cannot be instituted at the present time and should be reexamined when the cause(s) of the positive results are identified and corrected.

Nutrient composition and antimicrobial activity of sorrel drinks (soborodo).

Aqueous extracts (1,200 mL) of roselle calyx (40 g), fortified with either orange juice or pineapple juice as sweetener and lemon grass as flavorant (sorrel drink), were analyzed with regard to their mineral composition (Na, Fe, Zn, Cu, Pb, Mn, and Ca), vitamin C content, and sensory evaluation. While the medicinal potentials were determined with respect to their inhibitory effect on the growth of Bacillus sp., Pseudomonas aeruginosa, Lactobacillus sp., and Corynebacterium sp. The results revealed that the roselle extract fortified with orange juice had higher vitamin C content than did those fortified with pineapple juice, while those fortified with pineapple juice had the best general acceptability. Zn, Na, and Ca were generally high in all the drinks; however, fortification with either pineapple or orange juice reduced the mineral content of the roselle extract. However, Pb, Cu, and Mn (toxic metals) were not detected. The antimicrobial effect of the unfortified roselle extract was low against the entire organism; however, fortification with pineapple juice and lemon grass greatly enhanced the inhibition of the growth of those organisms. They all had their highest inhibitory effect on the growth of P. aeruginosa. In view of the high Zn, Ca, Fe, Na, and vitamin C content as well as the antimicrobial activity, this cheaply produced drink from purely local materials could serve as a good replacement for expensive carbonated drinks.

Response surface optimization of the critical medium components for the production of alkaline protease by a newly isolated Bacillus sp.

PURPOSE: Optimization of the fermentation medium for maximum alkaline protease production was carried out using a new strain, Bacillus Sp. PE-11. METHODS: The carbon source (glucose), the nitrogen source (peptone) and salt solution were selected to optimize. A 2(3 )full factorial composite experimental design and response surface methodology were used in the design of experiments and in the analysis of results. This procedure limited the number of actual experiments performed while allowing for possible interactions between the three components. RESULTS AND DISCUSSION: The optimum values for the tested variables for the maximum alkaline protease production were; glucose 7.798 (g/L), peptone 9.548 (g/L) and salt solution 8.757%. The maximum alkaline protease production was 4,98,123 PU/L. This method was efficient; only 20 experiments were necessary to assess these conditions, and model adequacy was very satisfactory, as the coefficient of determination was 0.941. CONCLUSIONS: In the work, we have demonstrated the use of a central composite factorial design by determining the conditions leading to the high yield of enzyme production. Thus, smaller and less time consuming experimental designs could generally suffice for the optimization of many fermentation processes.