Spore Yield Research Articles

Functional and Quality Assessment of a Spore Harvester for Entomopathogenic Fungi for Biopesticide Production

The Green Revolution led to an increased use of synthetic pesticides, causing environmental pollution. As an alternative, biopesticides made from entomopathogenic agents such as fungi have been sought. This study aimed to design and evaluate the performance of a harvester machine for efficiently collecting entomopathogenic spores of Metarhizium anisopliae and Beauveria bassiana grown on rice and corn substrates. The spore yield was estimated, and a spore count and a colony-forming unit (CFU) count were performed. Statistical analysis was conducted to compare the mean values of spores obtained from different combinations of solid substrate and fungi. The Corn-Metarhizium combination produced 34.15 g of spores per kg of substrate and 1.51 × 109 CFUs mL−1. Similarly, the Rice-Metarhizium combination produced 57.35 g per kg and 1.59 × 109 CFUs mL−1. Meanwhile, the Corn-Beauveria combination yielded 35.47 g per kg and 1.00 × 109 CFUs mL−1, while the Rice-Beauveria combination had a yield of 38.26 g per kg and 4.50 × 108 CFUs mL−1. Based on the reported results, the Rice-Metarhizium combination appears to be the most effective, yielding the highest number of harvested spores per kg of substrate. The study estimated a total cost of approximately $409.31 for manufacturing the harvester, considering only the cost of the materials. These results could potentially increase the availability and affordability of entomopathogenic fungi in integrated pest management.

Investigation of a Broad-Bean Based Low-Cost Medium Formulation for Bacillus subtilis MSCL 897 Spore Production

Bacillus subtilis (Bs) is a bacterium that benefits plants and is used in the production of bio-fungicides. The cultivation of Bs is a crucial step in bio-control preparation production, as it greatly impacts the quality and price of the final product. In a series of shake flask experiments, we investigated the economically feasible broth composition for spore production of Bacillus subtilis MSCL 897, a Latvian soil isolate. Our study investigated the impact of utilizing legume-based flours (such as broad bean, grey pea, and soybean) as the primary nitrogen source, along with sugar-beet molasses, sucrose, or glucose as the carbon source, and yeast extract, peptone, and corn-steep liquor as growth factor additives. Additionally, we examined the effect of using (NH3)2HPO4 or urea as supplementary nitrogen sources, as well as previously established media formulations, on spore yield. Our results showed that a culture medium composed of broad bean flour (10 g/L) and molasses (10 g/L) led to spore productivity of 1.35 ± 0.47 × 108 CFU/mL at 48 h. By enriching the culture medium base constituents with a minor (0.5–1.0 g/L) yeast extract or corn-steep liquor additive, a notable increase in spore productivity was observed, with values of 2.00 ± 0.28 × 108 and 2.34 ± 0.18 × 108 CFU/mL at 48 h, respectively, and sporulation efficiency > 80–90%. As a result, we achieved a high spore yield of the Bacillus subtilis MSCL 897 strain, demonstrating the competitiveness of our approach, which relied on a low-cost medium made mainly from locally available and renewable raw materials.

Infectivity of the parasite Metschnikowia bicuspidata is decreased by time spent as a transmission spore, but exposure to phycotoxins in the water column has no effect

Abstract Transmission from one host to another is a crucial component of parasite fitness. For some aquatic parasites, transmission occurs via a free‐living stage that spends time in the water, awaiting an encounter with a new host. These parasite transmission stages can be impacted by biotic and abiotic factors that influence the parasite's ability to successfully infect or grow in a new host. Here we tested whether time spent in the water column and/or exposure to common cyanobacterial toxins impacted parasite transmission stages. More specifically, we tested whether the infectivity, within host growth, and virulence of the fungal parasite Metschnikowia bicuspidata changed as a result of time spent in the water or from exposure to cyanotoxins in the water column. We exposed parasite transmission spores to different levels of one of two ecologically important cyanotoxins, microcystin‐LR and anatoxin‐a, and factorially manipulated the amount of time spores were incubated in water. We removed the toxins and used those same spores to infect one genotype of the common lake zooplankton Daphnia dentifera. We found that cyanotoxins did not impact parasite fitness (infection prevalence and spore yield per infected host) or virulence (host lifetime reproduction and survivorship) at the tested concentrations (10 and 30 μg/L). However, we found that spending longer as a transmission spore decreased a spore's chances for successful infection: spores that were only incubated for 24 hr infected approximately 75% of exposed hosts, whereas spores incubated for 10 days infected less than 50% of exposed hosts. We also found a negative relationship between the final spore yield from infected hosts and the proportion of hosts that became infected. In treatments where spores spent longer in the water column prior to encountering a host, infection prevalence was lower (indicating lower per spore infectivity), but each infected host yielded more spores at the end of infection. We hypothesise that this pattern may result from intraspecific parasite competition within the host. Overall, these results suggest that transmission spores of this parasite are not strongly influenced by cyanotoxins in the water column, but that other aspects of spending time in the water strongly influence parasite fitness.

Natural variation in host feeding behaviors impacts host disease and pathogen transmission potential.

Animals ranging from mosquitoes to humans often vary their feeding behavior when infected or merely exposed to pathogens. These so-called "sickness behaviors" are part of the innate immune response with many consequences, including avoiding orally transmitted pathogens. Fully understanding the role of this ubiquitous behavior in host defense and pathogen evolution requires a quantitative account of its impact on host and pathogen fitness across environmentally relevant contexts. Here, we use a zooplankton host and fungal pathogen as a case study to ask if infection-mediated feeding behaviors vary across pathogen exposure levels and natural genetic variation in susceptibility to infection. Then, we connect these changes in behavior to pathogen transmission potential (spore yield) and fitness and growth costs to the host. Our results validate a protective effect of altered feeding behavior during pathogen exposure while also revealing significant variation in the magnitude of this response across host susceptibility and pathogen exposure levels. Across all four host genotypes, feeding rates were negatively correlated with susceptibility to infection and transmission potential. The most susceptible genotypes exhibited either strong anorexia, reducing food intake by 26%-42%, ("Standard") or pronounced hyperphagia, increasing food intake by 20%-54% ("A45"). Together, these results suggest that infection-mediated changes in host feeding behavior-which are traditionally interpreted as immunopathology-may in fact serve as crucial components of host defense strategiesandwarrantfurtherinvestigation.

Spore yield of the microsporidium Nosema pyrausta (Paillot) Weiser, 1961 is influenced the insect host rearing temperature and diet composition

Spore yield of the microsporidium Nosema pyrausta (Paillot) Weiser, 1961 is influenced the insect host rearing temperature and diet composition

Breeding of Highly Virulent Beauveria bassiana Strains for Biological Control of the Leaf-Eating Pests of Dalbergia odorifera

Dalbergia odorifera (D. odorifera), commonly named the fragrant rosewood, is one of the second-level protected wild plants in China, and one of 34 species of rosewood in five genera and eight categories in the National Standard of China. As a kind of traditional Chinese medicine (TCM), it plays an important role in the pharmaceutical industry, including the treatment of cardiovascular diseases, rheumatic pain, etc. With the continuous expansion of the planting area of D. odorifera, the diseases and pests of D. odorifera become more and more serious, among which leaf-eating pests are the most serious. In this study, ultraviolet rays and microwaves were used to mutagenize Beauveria bassiana (B. bassiana) strain HNCMBJ-P-01, and excellent mutant strains with high spore yield and high virulence were screened out, and then they were prepared into a wettable powder for forest control experiments to study their biocontrol effects. The virulence screening test showed that the virulence of strain HBWB-44 was the strongest, and the 10 day corrected mortality rate was 80.00%, and the lethal time was 5.622 days. The results of biological control test showed that the control effect of B. bassiana wettable powder 100 times solution reached 60.89%, second only to the botanical fungicide matrine. Generically, The B. bassiana that we screened and mutated showed a good killing effect on Plecoptera bilinealis (P. bilinealis), and the wettable powder produced by it showed a good control effect on the leaf-eating pests of D. odorifera. The application of fungal insecticides in plantations has a good prospect for controlling the occurrence of leaf-eating pests of D. odorifera.

Sensitivity baseline establishment and resistance risk assessment of Botrytis cinerea from Panax ginseng to prochloraz

This study aimed to establish the baseline sensitivity of Botrytis cinerea from Panax ginseng to prochloraz, and ensure the fitness of prochloraz-resistant mutants and the cross-resistance of B. cinerea to prochloraz and commonly used fungicides for the prevention and control of gray mold including boscalid, pyraclostrobin, iprodione, and pyrimethanil. The sensitivity of B. cinerea from P. ginseng to fungicides was determined by the mycelial growth rate method. The prochloraz-resistant mutants were screened out through fungicide domestication and ultraviolet(UV) induction. The fitness of resistant mutants was determined through the stability of subculture, mycelial growth rate, and pathogenicity test. The cross-resistance between prochloraz and the four fungicides was determined by Person correlation analysis. The results showed that all B. cinerea strains tested were sensitive to prochloraz, and the EC_(50) value ranged from 0.004 8 to 0.062 9 μg·mL~(-1), with an average of 0.022 μg·mL~(-1). The sensitivity frequency distribution diagram showed that 89 B. cinerea strains were located within the main peak with a continuous single peak curve, and the average EC_(50) value of 0.018 μg·mL~(-1) was taken as the baseline sensitivity of B. cinerea to prochloraz. The fungicide domestication and UV induction obtained 6 resistant mutants, among which 2 strains were unstable and the other 2 strains showed decreased resistance after multiple generations of culture. Furthermore, the mycelial growth rate and spore yield of all resistant mutants were lower than those of their parents, and the pathogenicity of most mutants was lower than that of their parents. In addition, prochloraz had no obvious cross-resistance with boscalid, pyraclostrobin, iprodione, and pyrimethanil. In conclusion, prochloraz has great potential for controlling gray mold in P. ginseng, and the resistance risk of B. cinerea to prochloraz is low.

Comparison of one-step with two-step production of Bacillus atrophaeus spores for use as bioindicators.

The production method of spores significantly influences the resistance of spores used as bioindicators (BI) in the validation of sterilization of packaging material surfaces in aseptic food manufacturing. Therefore, the standardization of the spore production method represents an important and desirable goal in industrial BI production to ensure reliable validation test results. Previously, we recommended a two-step production approach for submerged spore production, in which the cultivation phase to obtain high cell mass was separate from the sporulation phase. In this work, a one-step manufacturing process was investigated to reduce production complexity and facilitate standardization of spore production. It was found that one-step BI production is technically possible but at the expense of spore yield. The two-step manufacturing process can realize almost 10-fold higher spore yields.

Enhancement of antibacterial and growth-promoting effects of Paenibacillus polymyxa by optimizing its fermentation process.

We aimed to enhance the antibacterial and growth-promoting effects of Paenibacillus polymyxa by improving the yield of spores, lipopeptides and indole-3-acetic acid (IAA) in the fermentation process. Through medium optimization by the response surface method and feeding fermentation, the number of spores reached 2.37 × 109 cfuml-1 with an increase of 38%, the content of lipopeptides reached 60.8mg L-1 with an increase of 89%, and the content of IAA reached 24.3mg L-1 with an increase of 176%, respectively, comparing with the original (un-optimized) culture conditions. The fermentation culture of P. polymyxa from the optimized medium and feeding fermentation resulted in higher colonization of P. polymyxa in soils than that from the original culture during the 49 days for testing. Comparing with the supernatant of the original culture, the supernatant of the P. polymyxa culture from the optimized medium and feeding fermentation showed enhanced antibacterial effects and plant growth-promoting effects. The enhanced antibacterial effect was shown as the increase of the inhibition zone by 59%, 45% and 26% against Ralstonia solanacearum, Erwinia carotovora and Xanthomonas campestris. The enhanced growth-promoting effects on tomato and strawberry plants were the increase of plant height by 47% and 5%, root length by 23% and 15% and root weight by 65% and 110%. The combination of medium optimization and feeding fermentation effectively improved the yield of spores, lipopeptides and IAA. Lipopeptides and IAA lead to enhanced antibacterial and plant growth-promoting effects of the P. polymyxa product. The optimized fermentation method significantly improved the yield of spores, lipopeptides and IAA, thus providing theoretical and technical support for enhancing the antibacterial and growth-promoting effects of P. polymyxa products in agriculture.

S6.3c Adaptive dynamics in experimental populations of Aspergillus nidulans

S6.3 Fungal adaptation and evolution, September 22, 2022, 4:45 PM - 6:15 PM A total of 12 replicate populations initiated with a laboratory strain of the ascomycete fungus Aspergillus nidulans evolved on synthetic minimal glucose agar medium for 1 year, using weekly transfers of 1% of the produced asexual spores to fresh medium. This Aspergillus short-term evolution experiment (ASEX) was designed to understand how filamentous fungi adapt to growth on limited carbon in a spatially structured environment. We observed no systematic improvement in the fitness components tested and neither in the competitive fitness relative to the ancestor. Instead, we observed the repeated evolution of at least two morphotypes, with a fluffy-like (FL) or an ancestor-like (AL) colony morphology, leading to non-transitive fitness interactions among isolates in two selected populations. The genomic analyses of clones from all 12 populations at an early (week 10) and the final time point (week 52), show a clear role of natural selection during ASEX. We also observed a shared genetic basis and different timing of adaptation of AL and FL types. In addition, in most populations, both morphotypes do not form monophyletic groups, but they frequently disappear and re-evolve from ancestral forms of both types. Reduction in asexual spore yield, the most evident parallel phenotypic change found in all our evolved populations, is not due to the direct selection of genes involved in asexual reproduction. Instead, we argue that reduced spore yield is a pleiotropic effect of adaptive changes in metabolism.

AMPK Is Involved in Regulating the Utilization of Carbon Sources, Conidiation, Pathogenicity, and Stress Response of the Nematode-Trapping Fungus Arthrobotrys oligospora.

ABSTRACTAMP-activated protein kinase (AMPK), a heterotrimeric complex, can sense energy and nutritional status in eukaryotic cells, thereby participating in the regulation of multiple cellular processes. In this study, we characterized the function of the catalytic α-subunit (SNF1) and the two regulatory β- and γ-subunits (GAL83 and SNF4) of AMPK in a representative nematode-trapping fungus, Arthrobotrys oligospora, by gene knockout, phenotypic analysis, and RNA sequencing. The ability of the AMPK complex mutants (including ΔAosnf1, ΔAogal83, and ΔAosnf4) to utilize a nonfermentable carbon source (glycerol) was reduced, and the spore yields and trap formation were remarkably decreased. Moreover, AMPK plays an important role in regulating stress response and nematode predation efficiency. Transcriptomic profiling between the wild-type strain and ΔAosnf1 showed that differentially expressed genes were enriched for peroxisome, endocytosis, fatty acid degradation, and multilipid metabolism (sphingolipid, ether lipid, glycerolipid, and glycerophospholipid). Meanwhile, a reduced lipid droplet accumulation in ΔAosnf1, ΔAogal83, and ΔAosnf4 mutants was observed, and more vacuoles appeared in the mycelia of the ΔAosnf1 mutant. These results highlight the important regulatory role of AMPK in the utilization of carbon sources and lipid metabolism, as well as providing novel insights into the regulatory mechanisms of the mycelia development, conidiation, and trap formation of nematode-trapping (NT) fungi.IMPORTANCE NT fungi are widely distributed in various ecosystems and are important factors in the control of nematode populations in nature; their trophic mycelia can form unique infectious devices (traps) for capturing nematodes. Arthrobotrys oligospora is a representative NT fungi which can develop complex three-dimensional networks (adhesive networks) for nematode predation. Here, we demonstrated that AMPK plays an important role in the glycerol utilization, conidiation, trap formation, and nematode predation of A. oligospora, which was further confirmed by transcriptomic analysis of the wild-type and mutant strains. In particular, our analysis indicated that AMPK is required for lipid metabolism, which is primarily associated with energy regulation and is essential for trap formation. Therefore, this study extends the functional study of AMPK in NT fungi and helps to elucidate the molecular mechanism of the regulation of trap development, as well as laying the foundation for the development of efficient nematode biocontrol agents.

Optimization of Sporulation Conditions for Bacillus subtilis BSNK-5

Bacillus subtilis spores have important biological applications; however, high spore-cell densities and sporulation efficiencies in fermentation is poorly reported. This study systematically analyzed the spore densities and formation efficiency of B. subtilis BSNK-5 in different culture substrates. A response surface regression equation was established based on the results of single factor and Box–Behnken experimental designs. The optimal medium formulation, as predicted from the equation, consisted of soluble starch at 3 g·L−1, soybean flour at 12 g·L−1, and MgSO4 at 5 g·L−1. The spore yield reached 2.43 × 109 CFU·mL−1, and the sporulation rate was 83.3%, which was nearly three times higher than before optimization using an optimized medium at 36 °C and 200 rpm for 60 h.

Optimization of sporulation and purification methods for sporicidal efficacy assessment on Bacillus spores.

Validating the efficacy of sporicidal agents is a critical step in current good manufacturing practices for disinfection requirements. A limitation is that the poor quality of spores can lead to false positive sporicidal results. The aim of this study was to explore optimal sporulation and purification methods in Bacillus spores. Spores of 7 Bacillus strains were produced in 5 different sporulation media. After density centrifugation, spore yields were measured by phase-contrast microscopy and enumeration assays. Effects of purification methods including heat, sonication and lysozyme, and maturation on spore qualities were determined by sodium hypochlorite sporicidal assay. Difco sporulation media was identified as the preferred sporulation medium for 4 out of 7 tested Bacillus strains. Sporulation rates in B. cereus, B. sphaericus, and B. thuringiensis were higher at 30°C than the rates at 37°C at a difference of 5%, 65%, and 20%, respectively. Bacillus licheniformis favored Mn2+-amended 10% Columbia Broth at 37°C for sporulation with 40-72% higher sporulation rates than other media. The maximum sporulation rates of B. cereus and B. thuringiensis were observed on double-strength Schaeffer's-glucose broth. All studied purification methods improved the spore purity with strain variations. However, intense heat (80°C for 20min) and lysozyme (100μg/mL) treatment impaired the spore quality of specific Bacillus strains by sensitizing them against sodium hypochlorite. The length of the maturation period had an impact on the spore resistance, and the most optimal maturation periods ranged from 7 to 21 days in Bacillus strains. The results of this study will pave the way for further evaluation of the sporicidal activity of disinfectants.

Virulence evolution during a naturally occurring parasite outbreak

Virulence, the degree to which a pathogen harms its host, is an important but poorly understood aspect of host-pathogen interactions. Virulence is not static, instead depending on ecological context and potentially evolving rapidly. For instance, at the start of an epidemic, when susceptible hosts are plentiful, pathogens may evolve increased virulence if this maximizes their intrinsic growth rate. However, if host density declines during an epidemic, theory predicts evolution of reduced virulence. Although well-studied theoretically, there is still little empirical evidence for virulence evolution in epidemics, especially in natural settings with native host and pathogen species. Here, we used a combination of field observations and lab assays in the Daphnia-Pasteuria model system to look for evidence of virulence evolution in nature. We monitored a large, naturally occurring outbreak of Pasteuria ramosa in Daphnia dentifera, where infection prevalence peaked at ~ 40% of the population infected and host density declined precipitously during the outbreak. In controlled infections in the lab, lifespan and reproduction of infected hosts was lower than that of unexposed control hosts and of hosts that were exposed but not infected. We did not detect any significant changes in host resistance or parasite infectivity, nor did we find evidence for shifts in parasite virulence (quantified by host lifespan and number of clutches produced by hosts). However, over the epidemic, the parasite evolved to produce significantly fewer spores in infected hosts. While this finding was unexpected, it might reflect previously quantified tradeoffs: parasites in high mortality (e.g., high predation) environments shift from vegetative growth to spore production sooner in infections, reducing spore yield. Future studies that track evolution of parasite spore yield in more populations, and that link those changes with genetic changes and with predation rates, will yield better insight into the drivers of parasite evolution in the wild.

Rapid and mass production of biopesticide Trichoderma Brev T069 from cassava peels using newly established solid-state fermentation bioreactor system

Converting agricultural waste into value-added biopesticides to replace chemical pesticides for plant protection is a good alternative for environmental sustainability and resource recycling. In this study, five tropical wastes (cassava peels, banana pseudostem, coconut shell, sugarcane bagasse, and pineapple peels) were screened as substrates for the rapid production of biopesticide Trichoderma Brev T069. Five single tests and a Box-Behnken design (BBD) with response surface methodology were used to optimize the culture conditions to improve the spore yield. The results showed that cassava peel was the optimal solid fermentation substrate, and the optimization enabled a spore yield of 9.31 × 109 spores/g at 3rd day, which was equal to 93.19% of spore yield obtained at 5th day (9.99 × 109 spores/g). A newly packed-bed bioreactor with agitation and ventilation system was developed and used to expand the production that 250 kg of biopesticide (2.89 × 109 spores/g) could be available on the 3rd day. A pot experiment indicated that the biopesticide T. Brev T069 obtained under this production system, when applied at 1 × 107 spores/g of soil had a 64.65% biocontrol efficiency on banana fusarium wilt. This study provides a practical solution for turning a tropical waste into an effective biopesticide which can prevent banana wilt disease, thereby helping to reduce disease management cost and overcome environmental hazards caused by synthetic pesticides.

Optimization of an economical medium composition for the coculture of Clostridium butyricum and Bacillus coagulans

Clostridium butyricum is a spore-forming probiotic which can promote the enhancement of beneficial bacteria and maintain intestinal microecological balance. However, it is difficult to improve the production level of C. butyricum by conventional fermentation process. In this study, a co-fermentation process of C. butyricum DL-1 and Bacillus coagulans ZC2-1 was established to improve the viable counts and spore yield of C. butyricum, and the formula of coculture medium was optimized by flask fermentation. The results showed that the optimum medium composition is 10 g/L bran, 15 g/L corn steep powder, 15 g/L peptone, 1 g/L K2HPO4 and 0.5 g/L MnSO4.Cultured stationarily in the optimal medium for 36 h, the number of viable bacteria of C. butyricum DL-1 reached 1.5 × 108 cfu/mL, Which was 375 times higher than that incubated in the initial medium. The sporulation rate reach 92.6%. The results revealed an economical and effective medium composition for the coculture of C. butyricum and B. coagulans, which achieved a 64.6% cost reduction. The co-fermentation process established in this study provides a new fermentation mode for the industrial production of other absolute anerobic bacteria.

Multiple linear regression models to simulate spore yields of Bacillus amyloliquefaciens BS13 through optimization of medium composition.

Bacillus amyloliquefaciens is a food spoilage spore-forming bacterium. Its spores are useful for multiple biotechnological applications. Nevertheless, few reports are available regarding the achievement of a high cell density and good sporulation effectiveness under fermentation conditions. Therefore, the current study was designed to optimize a low-cost fermentation medium allowing the highest sporulation yield by B. amyloliquefaciens strain BS13. Our data revealed that tryptone and starch were the best carbon and energy sources. In addition, two nitrogen sources namely, corn steep liquor (CSL) and yeast extract (YE), allowed a significant enhancement of spore production and they were both retained for further optimization. A combination of CaCl2 , MgSO4 , and MnSO4 showed a positive impact on spores' production. The composition of the optimized medium was (in g/L); tryptone 3, starch 15, CSL 13.5, YE 1.5, CaCl2 0.1, MgSO4 ·7H2 O 0.012, and MnSO4 ·7H2 O 0.0012. Such medium was further validated in a 400-L fermentor. The spore yield by B. amyloliquefaciens strain BS13 was enhanced from 3.0 × 1010 spores/mL under flask culture conditions to 6.2 × 1010 spores/mL when cultures were performed on large scale. Therefore, strain BS13 spore preparation could be proposed as a promising probiotic and a biocontrol agent useful for plants, animals, and humans.

Optimization of Growth Conditions for the Production of Bacillus subtilis Using Central Composite Design and Its Antagonism Against Pathogenic Fungi.

Today, the enhancement of spore yields of Bacillus subtilis has considerable interest and has been widely investigated. In this context, studies have been carried out to improve the spore yield as well as the production amount. In order to perform this, optimization studies are conducted for large-scale production of B. subtilis in bioreactors. The prokaryotic structure, high extracellular production potential, lack of pathogenic activity, well-known fermentation technology and short fermentation time are the prominent advantages for the production of B. subtilis in a bioreactor. The Bacillus species produce a wide variety of antifungal and antimicrobial compounds, making them ideal biological control agents. In this study, first, the growth conditions of the medium were investigated and then optimized using the central composite design approach to achieve the highest productivity for the growth of B. subtilis. In the experiments, the effect of temperature of 25, 30 and 35°C and pH level of 6.0, 7.0 and 8.0 on spore yield was studied. Moreover, the antifungal activity of the B. subtilis culture was investigated against pathogenic fungi: Colletotrichum gloeosporioides, Botrytis cinerea and Aspergillus brasiliensis.

Biotechnological potential of Zymotis-2 bioreactor for the cultivation of filamentous fungi.

A new prototype of Solid-State Fermentation Bioreactor, namely "Zymotis-2 ", was developed to produce fungal spores. A fermentation process for fungal spores, and hydrolase enzymes (endo and exoglucanases, amylases) production by Trichoderma asperellum DWG3, Aspergillus niger G131 and Beauveria bassiana was scaled-up from flasks and glass Raimbault column packed with 20g of solid substrates (dry weight) to 5kg of solid substrate by using the new Zymotis-2 bioreactor. Fungi strains growth using a mix of vine shoots, wheat bran, and olive pomace was tested under similar experimental conditions in Zymotis-2 bioreactor, column bioreactor and flasks in a parallel fermentation system. Overall, significant spore production on Zymotis-2 bioreactor was obtained, achieving 22.01 ±0.01×109 spores/g DM 16.30 ±0.07 × 109 spores/g DM, and 3.30 ±0.07 × 109 spores/g DM for B. bassiana, T. asperellum DWG3, and A. niger G131, respectively. Forced aeration increased the endoglucanases, exoglucanases and amylases activities for T. asperellum DWG3 but B. bassiana and A. niger G131 were affected negatively by the aerated process, showing the lowest enzyme activities. In conclusion, a high yield of spores was obtained at 137 h of cultivation time, confirming the validity of the new Zymotis-2 bioreactor to produce virulent spores at low cost by T. asperellum, B. bassiana and A. niger G131.

Probiotic Tofu with Bacillus clausii Spores to Support Gastrointestinal Microflora

Objective: This study was conducted to develop a tofu product enriched with probiotic Bacillus clausii spores that vegan individuals can benefit from without changing their diets for probiotic consumption and without having to use the supplementary probiotic pills.Method: B. clausii was cultured to sporulate in 2xSG medium. The resulting B. clausii spores were added to tofu by two different methods to determine the optimum step in the procedure to ensure that the probiotic was able to retain in tofu at the highest amount; i) addition of probiotic spores before coagulant MgCl2 (P-C tofu), ii) addition of probiotic spores after the coagulant (C-P tofu).Results: The yields of probiotic spores in P-C and C-P tofu were calculated to be 5.45±0.40 and 5.33±0.56 log cfu/g, respectively, which was slightly lower than the targeted level, 6 log cfu/g. No significant difference was observed between the P-C and C-P tofu methods in terms of probiotic concentration (P=0.36; t test). Storage of P-C tofu at -18°C decreased the probiotic load by 0.45 log cfu/g in 1 month (P=0.003). One-day temperature abuse at 20°C of cold chain storage resulted in a significant 0.85 log reduction (P=0.01). Sauté cooking was found to decrease the spore load in tofu significantly by 1.22 log (P=0.004). Sensory evaluation of P-C and probiotic free tofu did not reveal a significant difference in general acceptability features (P>0.05). Conclusion: Probiotic enriched tofu may be a healthy alternative choice for vegans and vegetarians while positively affecting the health of consumers.