Understanding the mechanism of interaction of candidate soil stabilizing prototypes by using microscopy and spectroscopy techniques.

Globally, there is a high demand for bio-based soil stabilizers required for improving the strength properties of weak in situ soil. Microbes and microbial components such as Bacillus spp. have gained interest as soil stabilizers due to their production of spores, bio-enzymes, and bio-polymers. However, the current approach for any microlevel assessment of bio-additives and in situ soil improvement is limited. This paper provides data for microstructural evaluation of stabilized soil material for the postulation of the mode of action. In this study, the microbonding effect (i.e., bio-based cementation, bio-clogging, and soil particle bio-coating) is successfully observed within the various stabilizing prototypes, obtained from a novel Bacillus spp. using advanced methods, namely field emission gun-scanning electron microscopy and Fourier transform-infrared spectroscopy. The results show that treated soil versus untreated soil properties are altered by the bio-additive/s stabilizing effect. These indicator tests provide data for further bio-stabilizer product prototype development and processes (i.e., improved products in terms of strength and moisture susceptibility). The use of microscopy and spectroscopy was sufficient for the preliminary selection of suitable candidates for soil stabilization.

Production and stability of a multi-strain Bacillus based probiotic product for commercial use in poultry.

Probiotics can be effective alternatives to the prophylactic use of antibiotic growth promoters (AGPs) in response to industry and consumer concerns around their use in poultry. Studies on the suitability of Bacillus probiotics are emerging and showing benefits, but information on the production technology is limited. We developed the production process for a novel probiotic product previously shown to be effective in field trials. All strains were cultivated to a spore concentration exceeding 1 × 1010 CFU. mL-1. The spores of each strain were harvested, processed into a powder intermediate and formulated into an end product with 100 % recoveries and a shelf life stability >1 year. The probiotic was shown to be incorporated into broiler feed exceeding the desired concentration of 1 × 106 CFU. g-1. Using efficient process technology and lower cost materials, this study presents a commercially relevant case for the potential adoption of probiotic products by the poultry industry.

Isolation, selection and evaluation of Bacillus spp. as potential multi-mode probiotics for poultry.

Bacillus based probiotics are becoming relevant as alternatives to antibiotics used in poultry production and in other animal husbandry. This study describes the isolation of 48 Bacillus spp. candidates, from chickens and chicken environments, for use as potential probiotics in poultry production. These isolates, plus a further 18, were tested in a comprehensive in vitro screening regime that was specifically designed to select the best isolates that satisfied multiple modes of action desirable for commercial poultry probiotics. This screening programme involved the evaluation of the ability of the isolates to survive and grow in the limiting conditions of the chicken gastrointestinal tract. Only 11 of the isolates fulfilled these criteria; hence, they were further evaluated for the ability to adhere to epithelial cells, produce extracellular enzymes, and to demonstrate antagonistic activity against selected pathogens of significant importance in poultry production. Of these, a total of 6 isolates were selected, due to their all-round probiotic capability. Identification by 16S RNA sequencing confirmed these isolates as B. subtilis and B. velezensis, identities which are generally regarded as safe. The Bacillus isolates reported in our study exhibit strong all-inclusive probiotic effects and can potentially be formulated as a probiotic preparation for poultry production.

A novel Bacillus based multi-strain probiotic improves growth performance and intestinal properties of Clostridium perfringens challenged broilers.

There is a necessity for the implementation of in-feed probiotics in the poultry production industry, following strict regulations around the use of antibiotic growth promoters (AGP). Bacillus spp. are becoming an attractive alternative because of their functionality and stability. This study aims to evaluate the effect of a novel multi-strain Bacillus based probiotic on growth performance and gut health in male Ross 308 broiler chickens challenged with Clostridium perfringens Type A. Broilers on a 4 phase feeding program were fed diets containing either a standard metabolizable energy (ME) (100%) or a reduced ME (98%) level. The test probiotic was compared to an un-supplemented negative control and a commercial benchmark product as positive control over a 35 D feeding trial, using a 2 × 3 factorial experimental design. Chicks were inoculated with a once-off dose of C. perfringens on day 14. Growth performance was measured weekly to calculate body weight (BW), feed intake (FI) and feed conversion ratio (FCR). Villi histomorphology, gut lesions, and liver weight were assessed at day 35. Broilers fed the reduced ME diet with the test probiotic achieved higher final BWs (P = 0.037) and FCR (P = 0.014) than the negative control. Broilers fed the standard ME diet with the test probiotic showed improved (P = 0.001) FCR than the negative control from day 21 onwards. Increased duodenal villi height (P = 0.012) and villi height to crypt depth ratio in the duodenum (P < 0.0001) and jejunum (P = 0.0004) were observed in broilers fed the reduced ME diet containing the test probiotic. Additionally, the test probiotic resulted in significantly reduced relative liver weights in both ME groups. Consequently, the results suggest that the novel multi-strain Bacillus based probiotic enhanced broiler performance and improved gut health and is thus attractive as an alternative to AGP's in broiler production.

Establishing miniaturised structural testing techniques to enable high-throughput screening of microorganisms and microbial components for unpaved road stabilisation application.

Roads are expensive to develop particularly in challenging environmental conditions, and a lack of understanding of the properties of soil can lead to poor design and premature failures contributing to costly maintenance. The construction industry is exploring new opportunities involving biological processes and products to modify the structural properties of the in situ material, in terms of strength, volume stability, durability and permeability. Through an integrative interdisciplinary approach several microorganisms and other existing bio-enzymatic products such as secondary metabolites, enzymes, endospores, and extracellular polymeric substances have been considered as possible alternatives to conventional methods for the development of sustainable road infrastructure. Limitations in the current state of technology to developing bio-based solutions include microorganism selection and the ability to evaluate derivative components in rapid structural tests that enhance the time to development of proper commercial products. This study focused on the testing of fermentation derived components of biological materials in a high-throughput manner, using miniaturised structural tests to validate screening and selection methodology. The methods tested included resistance to abrasion, resistance to erosion, water absorption and resistance to compression load. Unique miniaturised test equipment was successfully developed using computer-aided design (CAD) and 3D printing technologies. Effects were measured to enable the rapid evaluation of a target microorganism and for screening of biological components or fractions. Results obtained using a Bacillus isolate reported in the current study exhibit strength characteristics and can potentially be formulated as a product for soil stabilisation. This work forms the basis for in vitro selection methodology to enhance development of bio-based structural materials for application in the road sector.

A downstream process for production of a viable and stable Bacillus cereus aquaculture biological agent.

Biological products offer advantages over chemotherapeutics in aquaculture. Adoption in commercial application is lacking due to limitations in process and product development that address key end user product requirements such as cost, efficacy, shelf life and convenience. In previous studies, we have reported on the efficacy, physiological robustness and low-cost spore production of a Bacillus cereus isolate (NRRL 100132). This study examines the development of suitable spore recovery, drying, formulation and tablet production from the fermentation product. Key criteria used for such downstream process unit evaluation included spore viability, recovery, spore balance, spore re-germination, product intermediate stability, end product stability and efficacy. A process flow sheet comprising vertical tube centrifugation, fluidised bed agglomeration and tablet pressing yielded a suitable product. The formulation included corn steep liquor and glucose to enhance subsequent spore regermination. Viable spore recovery and spore balance closure across each of the process units was high (>70% and >99% respectively), with improvement in recovery possible by adoption of continuous processing at large scale. Spore regermination was 97%, whilst a product half-life in excess of 5 years was estimated based on thermal resistance curves. The process resulted in a commercially attractive product and suitable variable cost of production.

High-density spore production of a B. cereus aquaculture biological agent by nutrient supplementation.

Previous studies have demonstrated the efficacy of our Bacillus cereus isolate (NRRL 100132) in reducing concentrations of nitrogenous wastes and inhibiting growth of fish pathogens. In vivo efficacy and tolerance to a range of physiological conditions in systems used to rear Cyprinus carpio make this isolate an excellent candidate for aquaculture applications. Production cost is an important consideration in development of commercially relevant biological products, and this study examines the optimization of nutrient supplementation, which has an impact on high-density production of spores by fermentation. Corn steep liquor (CSL) was identified as a lower cost and more effective nutrient source in comparison to conventional nutrient substrates, in particular yeast extract and nutrient broth. The improved sporulation performance of B. cereus could be related to the increased availability of free amino acids, carbohydrates, and minerals in CSL, which had a positive effect on sporulation efficiency. The impact of nutrient concentration on spore yield and productivity was modeled to develop a tool for optimization of nutrient concentration in fermentation. An excellent fit of the model was confirmed in laboratory fermentation studies. A cost comparison revealed that production using liquid phytase and ultrafiltered-treated CSL was less expensive than spray-dried CSL and supported cultivation of B. cereus spores at densities higher than 1 x 10(10) CFU ml(-1).

Functionality of a Bacillus cereus biological agent in response to physiological variables encountered in aquaculture.

The potential of a Bacillus cereus isolate (NRRL 100132) as a biological agent for aquaculture has been demonstrated in vitro and in vivo. The functionality of this isolate across a range of physiological conditions, including salinity, pH and temperature, based on rearing of high-value ornamental Cyprinus carpio, was investigated. Temperature had a significant influence on germination, specific growth rate and increase in cell number of B. cereus in shake-flask cultures, whilst salinity and pH did not have a measurable effect on growth. Controlled studies in bioreactors and modelling of the data to the Arrhenius function indicated the existence of high and low growth temperature domains. The rates of pathogenic Aeromonas hydrophila suppression and decrease in waste ion concentrations (ammonium, nitrite, nitrate and phosphate) were translated into a linear predictive indicator of efficacy of the B. cereus isolate at different temperatures. The present study confirmed the robustness of the B. cereus isolate (NRRL 100132) as a putative biological agent for aquaculture and further demonstrated a novel method for the assessment of in vitro biological efficacy as a function of temperature.

Multi-copy expression and fed-batch production of Rhodotorula araucariae epoxide hydrolase in Yarrowia lipolytica.

Epoxide hydrolases (EHs) of fungal origin have the ability to catalyze the enantioselective hydrolysis of epoxides to their corresponding diols. However, wild type fungal EHs are limited in substrate range and enantioselectivity. Additionally, the production of fungal epoxide hydrolase (EH) by wild-type strains is typically very low. In the present study, the EH-encoding gene from Rhodotorula araucariae was functionally expressed in Yarrowia lipolytica, under the control of a growth phase inducible hp4d promoter, in a multi-copy expression cassette. The transformation experiments yielded a positive transformant, with a final EH activity of 220 U/g dw in shake-flask cultures. Evaluation of this transformant in batch fermentations resulted in approximately 7-fold improvement in EH activity over the flask scale. Different constant specific feed rates were tested in fed-batch fermentations, resulting in an EH activity of 1,750 U/g dw at a specific feed rate of approximately 0.1 g/g/h, in comparison to enzyme production levels of 0.3 U/g dw for the wild type R. araucariae and 52 U/g dw for an Escherichia coli recombinant strain expressing the same gene. The expression of EH in Y. lipolytica using a multi-copy cassette demonstrates potential for commercial application.