Spore Yield Research Articles

Study on Optimizing Nutrition and Fermentation Conditions for Compound <i>Bacillus spp.</i>

In order to improve the spore yield of compound Bacillus spp. (B. amyloliquefaciens, B. laterosporus and B. megaterium), the effects of nutrient conditions including carbon source, nitrogen source, mineral salt and fermentation conditions including the inoculum age, inoculation amount, loading volume of liquid and initial pH on the spore yield were studied. The results indicated that the optimized medium was glucoses 20 g/L, soybean meal 30.0 g/L, K2HPO4 1.0 g/L; fermentation temperature is 37℃, the inoculum age 12 h, initial pH 7.0, 2% inoculation amount, loading volume of liquid 20 mL/250 mL. Under the optimized conditions of culture medium and fermentation for compound Bacillus spp., spore yield was 10.24 times more than the initial medium, and the spore formation rate reached more than 90%.

An enhanced green fluorescence protein (EGFP)-based reporter assay for quantitative detection of sporulation in Clostridium perfringens SM101

Clostridium perfringens type F is a spore-forming anaerobe that causes bacterial food-borne illness in humans. The disease develops when ingested vegetative cells reach the intestinal tract and begin to form spores that produce the diarrheagenic C. perfringens enterotoxin (CPE). Given that CPE production is regulated by the master regulator of sporulation (transcription factor Spo0A), the identification of sporulation-inducing factors in the intestine is relevant to better understanding of the disease. To examine these factors, we established assays to quantify C. perfringens sporulation stage under microscopy by using two fluorescent reporters, namely, Evoglow-Bs2 and CpEGFP. When the reporter genes were placed under control of the cpe promoter, both protein products were expressed specifically during sporulation. However, the intensity of the anaerobic reporter Evoglow-Bs2 was weak and rapidly photobleached during microscopic observation. Alternatively, CpEGFP, a canonical green fluorescence protein with optimized codon usage for Clostridium species, was readily detectable in the mother-cell compartment of most bacteria at early stages of sporulation. Additionally, CpEGFP expression predicted final spore yield and was quantifiable in 96-well plates using fluorescence plate reader. These results indicate that CpEGFP can be used to analyze the sporulation of C. perfringens and has a potential application in the large-scale screening of sporulation-regulating biomolecules.

Multiplication of entomopathogenic fungus (Lecanicillium lecanii) on apple pomace and its toxicity against aphid (Aphis craccivora)

The apple pomace (AP) was standardized for the multiplication of Lecanicillium lecanii and evaluated against Aphis craccivora. Results showed that AP medium at 2% recorded more mycelial growth and were at par with AP 3%, 4%, and 5% as compared to other concentrations. The spore yield of L. lecanii also significantly higher in AP 5% (16 × 106 spores ml−1) and was at par with AP 0.5%. L. lecanii at 1 × 109 spores ml−1 showed maximum mortality (43.33–93.33% mortality) against A. craccivora and was followed by 1 × 108 spores ml-1 (36.67–86.67% mortality).

Bacterial Endospores as Phage Genome Carriers and Protective Shells.

Bacterial endospores can serve as phage genome protection shells against various environmental stresses to enhance microbial control applications. The genomes of polyvalent lytic Bacillus phages PBSC1 and PBSC2, which infect both B. subtilis subsp. subtilis and B. cereus NRS 248, were incorporated into B. subtilis endospores (without integration into the host chromosome). When PBSC1 and PBSC2 were released from germinating endospores, they significantly inhibited the growth of the targeted opportunistic pathogen B. cereus Optimal endospore entrapment was achieved when phages were introduced to the fast-sporulating prespores at a multiplicity of infection of 1. Longer endospore maturation (48 h versus 24 h) increased both spore yield and efficiency of entrapment. Compared with free phages, spore-protected phage genomes showed significantly higher resistance toward high temperatures (60 to 80°C), extreme pH (pH 2 or pH 12), and copper ions (0.1 to 10 mg/liter). Endospore germination is inducible by low concentrations of l-alanine or by a germinant mixture (l-asparagine, d-glucose, d-fructose, and K+) to trigger the expression, assembly, and consequent release of phage particles within 60 to 90 min. Overall, the superior resiliency of polyvalent phages protected by endospores might enable nonrefrigerated phage storage and enhance phage applications after exposure to adverse environmental conditions.IMPORTANCE Bacteriophages are being considered for the control of multidrug-resistant and other problematic bacteria in environmental systems. However, the efficacy of phage-based microbial control is limited by infectivity loss during phage delivery and/or storage. Here, we exploit the pseudolysogenic state of phages, which involves incorporation of their genome into bacterial endospores (without integration into the host chromosome), to enhance survival in unfavorable environments. We isolated polyvalent (broad-host-range) phages that efficiently infect both benign and opportunistically pathogenic Bacillus strains and encapsulated the phage genomes in B. subtilis endospores to significantly improve resistance to various environmental stressors. Encapsulation by spores also significantly enhanced phage genome viability during storage. We also show that endospore germination can be induced on demand with nutrient germinants that trigger the release of active phages. Overall, we demonstrate that encapsulation of polyvalent phage genomes into benign endospores holds great promise for broadening the scope and efficacy of phage biocontrol.

Effect of oil cakes and garlic aqueousbased formulations of trichoderma viride on management of meloidogyne incognita in chilli

Abstract Root-knot nematode, Meloidogyne incognita (Kofoid and White) is a major threat to chilli (Capsicum annum) cultivation, by forming root galls and subsequent wilting. These nematodes live in soil, roots debris and reported surviving in other Solanaceae crops. Chemical application of nematicides cause impact on environment, therefore biocontrol using antagonistic fungi is desired to tackle this problem. This research is aimed to evaluate the effect of Trichoderma viride and its formulations on management of M. incognita. Oil cakes such as neem (Azadiracta indica), gingelly (Sesamum indicum), Mahua (Madhuca longifolia) and garlic (Allium sativum) extracts were used to evaluate the growth and sporulation of T. viride. Chilli variety MI-2 was used. Among the four preparations, neem oil cake recorded high spore yield of 1.75 ×107 spores/ml. Similarly gingelly oil cake and garlic produced the spore yields of 1.57 ×107 spores/ml and 1.368 ×107 spores/ml, respectively. Comparatively Mahua oil cake obtained low spore yield of 7.87×106 spores/ml. Plant growth was significant (P<0.05) in the application of neem oil cake formulation (30.42 cm). Extent of galling was significantly low with garlic (9.00) and neem (9.25). These results confirmed that the formulations of neem, gingelly oil cake extracts and garlic aqueous extract stimulated the chilli plant growth, productivity and reduced the nematode infestation. Above all, neem oil cake and garlic are the best formulations that can be used to manage M. incognita

Isolation and characterization of Metarhizium anisopliae TK29 and its mycoinsecticide effects against subterranean termite Coptotermes formosanus

The entomopathogenic fungus Metarhizium anisopliae is widely used as biocontrol agent against many insect pests. In the present study, the potential isolate of M. anisopliae TK29 was isolated from the agricultural soils in Thekkady, India. The taxonomic identity of the isolate was confirmed based on its morphology and 18S rDNA gene sequence homology. Phylogenetic analysis confirmed that the isolated strains were related to the same species. A potential isolate (TK29) was optimized for mass cultivation and conidial spore production was enhanced using three different raw substrates (Rice, Maize, black gram) by solid-state fermentation. The results showed higher conidial spore yield from rice (2.6 ± 0.32%) compared to black gram (2.1 ± 0.28%) and maize (1.9 ± 0.23%) substrates. Dry green conidia were applied against Formosan subterranean termite, Coptotermes formosanus at three different concentrations (1 × 106, 1 × 107, and 1 × 108 conidia/ml−1). The highest mortality rate was obtained from 1 × 108 conidia/ml−1 at 120 h post-treatment. Our study indicated that M. anisopliae TK29 had desirable attributes for the development of a mycoinsecticide against C. formosanus.

Sporulation environment influences spore properties in Bacillus: evidence and insights on underlying molecular and physiological mechanisms

Bacterial spores are resistant to physical and chemical insults, which makes them a major concern for public health and industry. Spores help bacteria to survive extreme environmental conditions that vegetative cells cannot tolerate. Spore resistance and dormancy are important properties for applications in medicine, veterinary health, food safety, crop protection and other domains. The resistance of bacterial spores results from a protective multilayered structure and from the unique composition of the spore core. The mechanisms of sporulation and germination, the first stage after breaking of dormancy, and organization of spore structure have been extensively studied in Bacillus species. This review aims to illustrate how far the structure, composition and properties of spores are shaped by the environmental conditions in which spores form. We look at the physiological and molecular mechanisms underpinning how sporulation media and environment deeply affect spore yield, spore properties like resistance to wet heat and physical and chemical agents, germination and further growth. For example, spore core water content decreases as sporulation temperature increases, and resistance to wet heat increases. Controlling the fate of Bacillus spores is pivotal to controlling bacterial risks and process efficiencies in, for example, the food industry, and better control hinges on better understanding how sporulation conditions influence spore properties.

Utilization of Lignocellulose-based Orange Peel Waste for Induced Sporulation of Trichoderma asperellum via Box-Behnken Matrix Design

The feasibility of using orange peel residues as a substrate for induced sporulation of Trichoderma asperellum was evaluated. The Box-Behnken matrix (BBM) design was used to screen the effects of several parameters, including the effect of pH, inoculum, and moisture under solid-state fermentation culture conditions. The study was performed in two experimental steps (screening and optimization). Moisture content and pH were determined to be the most influential parameters on spore production during the screening stage. A Box-Behnken design was used to optimize and to define the interaction of the selected parameters. The moisture content was determined as the most significant parameter affecting spore production. An inoculum of 1 × 106 spores g-1, pH 6.07, and moisture content of 69.0% was the combination of conditions observed to achieve the maximum production of 2.04 × 109 spores g-1. The experimental value of 2.16 × 109 spores g-1 (from the experimental model) showed a good fit to the regressed model, with a standard error of 5%. Based on this work, a high yield of spores was obtained at 144 h of cultivation time, indicating that it is a feasible approach to use orange peel as a substrate for biomass and spore production.

Solid - state Fermentation Technology of Paecilomyces lilacinus and Development of Agricultural Biocontrol Agent Based on Agricultural Waste Resources

Studied the solid-state fermentation of low-cost Paecilomyces lilacinus by using mushroom residue, rice husk and other agricultural waste resources as the substrate. The results showed that the optimum solid fermentation medium includes the following components: mushroom residue: bran =1:1, soybean meal 1.5%, glucose 1.2%, KH2PO4 0.2%, MgSO4 0.08%, CaCO3 0.7%. The culture temperature was 28 °C, the loading thickness was 5 cm, the inoculation amount was 8%, the culture time was 7 days, and the spore yield could reach1.35×1010 cfu/g. The addition of 3 ‰ shrimp and crab shell powder, the fermentation product chitinase activity was significantly improved. The effect of adding 5 ‰ trehalose on the spore survival rate of Paecilomyces lilacinus increased by 89.3% by low-temperature vacuum drying. The work of this paper provides some guidance for the industrial production and application of Paecilomyces lilacinus.

Optimization of Solid-state Fermentation Process and Development of Biocontrol Agent Based on Cassava Residue from Streptomyces Microflavus

In this article, solid-state fermentation of low-cost Streptomyces microflavus is studied by using cassava residue, rice husk and other agricultural solid waste resources as the substrate to prepare biopharmaceuticals [1]. The experiment results showed that the Streptomyces microflavus’ optimum solid-state fermentation medium and conditions includes the following components: cassava residue 89.85%, rice husk 10%, KH2PO4 0.05%, NaCl 0.05%, MgSO4 0.05%. The fermentation temperature is controlled at about 30 °C, the initial water content is around 50%, initial pH is 6.6-7, the material thickness is 5cm, the inoculation amount is 7.5%, the cultivation time is 4 days, and the spore yield is 4.8*109 cfu/g. The survival rate of Streptomyces microflavus’ spores is 75%, which compared with blank conditions increased by 30% after low temperature vacuum drying. The work of this paper provides some theoretical guidance for the large-scale industrial production of Streptomyces, which has valuable application in agricultural field.

Rapid optimization of spore production from Bacillus amyloliquefaciens in submerged cultures based on dipicolinic acid fluorimetry assay

Some optimization techniques have been widely applied for spore fermentation based on the plate counting. This study optimized the culture medium for the spore production of Bacillus amyloliquefaciens BS-20 and investigated the feasibility of using a dipicolonic acid (DPA) fluorimetry assay as a simpler alternative to plate counting for evaluating spore yields. Through the single-factor experiment, the metal ions and agro-industrial raw materials that significantly enhanced spore production were determined. After conducting a response surface methodology (RSM) analysis of several metal ions, the combined use of optimum concentrations of Mn2+, Fe2+, and Ca2+ in culture media produced a 3.4-fold increase in spore yields. Subsequently, supplementing soybean meal and corn meal with optimum concentrations determined by another RSM analysis produced an 8.8-fold increase. The final spore concentration from a culture medium incorporating optimum concentrations of the metal ions and raw materials mentioned above was verified to reach (8.05 ± 0.70) × 109 CFU/mL by both DPA fluorimetry and plate counting. The results suggest that the use of DPA fluorescence intensity as an alternative value to colony counting provides a general method for assessing spore yields with less work and shorter time.

Bacillus amyloliquefaciens Spore Production Under Solid-State Fermentation of Lignocellulosic Residues.

This study was conducted to elucidate cultivation conditions determining Bacillus amyloliquefaciens B-1895 growth and enhanced spore formation during the solid-state fermentation (SSF) of agro-industrial lignocellulosic biomasses. Among the tested growth substrates, corncobs provided the highest yield of spores (47 × 1010 spores g-1 biomass) while the mushroom spent substrate and sunflower oil mill appeared to be poor growth substrates for spore formation. Maximum spore yield (82 × 1010 spores g-1 biomass) was achieved when 15g corncobs were moistened with 60ml of the optimized nutrient medium containing 10g peptone, 2g KH2PO4, 1g MgSO4·7H2O, and 1g NaCl per 1l of distilled water. The cheese whey usage for wetting of lignocellulosic substrate instead water promoted spore formation and increased the spore number to 105 × 1010 spores g-1. Addition to the cheese whey of optimized medium components favored sporulation process. The feasibility of developed medium and strategy was shown in scaled up SSF of corncobs in polypropylene bags since yield of 10 × 1011 spores per gram of dry biomass was achieved. In the SSF of lignocellulose, B. amyloliquefaciens B-1895 secreted comparatively high cellulase and xylanase activities to ensure good growth of the bacterial culture.

OPTIMIZATION OF ENHANCED PROBIOTIC SPORES PRODUCTION IN SUBMERGED CULTIVATION OF BACILLUS AMYLOLIQUEFACIENS B-1895

The aim of this study was to gain a deeper fundamental knowledge on the physiology of B. amyloliquefaciens B-1895 for development of probiotic production technology through the submerged fermentation of renewable and inexpensive agro-industrial lignocellulosic biomass. For comparison, bacilli cultivation was performed in the synthetic medium with different carbon sources. The highest yield of spores (7.1 x 109/mL) was attained after 72 h of submerged cultivation of bacilli in the 0.4% glucose-containing medium. Substitution of glucose with various lignocellulosic materials at concentration of 40 g/L yielded 2.3-10.8 x 109 spores/mL. After subsequent optimization, the developed simple medium containing milled corn cobs as a growth substrate and casein hydrolysate at concentration of 40 mM N provided formation of as high as 2.8 x 1010 spore/mL in shake-flasks experiments and 2.5 x 1010 spores/mL in a laboratory fermenter enabling the large scale production of low-cost probiotic for their biotechnological application. The study underlines importance for the efficient sporulation of carbon source depletion at the end of the exponential growth phase. Moreover, it shows that an exploitation of lignocellulosic materials with an appropriate chemical composition is a reasonable way to achieve high cell density and sporulation since bacilli secrete sufficient levels of glycosyl hydrolases for substrate saccharification to ensure bacterial culture with carbon and energy source.

Efficient Method for the Rapid Purification of Nosema ceranae Spores

Nosema ceranae is an obligate intracellular fungal parasite that causes mortality in honey bees and enhances the susceptibility of honey bees to other pathogens. Efficient purification of Nosema spores from the midgut of infected honey bees is very important because Nosema is non-culturable and only seasonably available. To achieve a higher yield of spores from honey bees, in this study, we considered that the initial release of spores from the midgut tissues was the most critical step. The use of 2 mm beads along with enzymatic treatment with collagenase and trypsin enhanced the homogenization of tissues and the yield of released spores by approximately 2.95 times compared with the use of common 3 mm beads alone. The optimal time for the enzyme treatment was determined to be 1 hr as measured by the yield and viability of the spores. A one-step filtration using a filter paper with an 8–11 μm pore size was sufficient for removing cell debris. This method may be useful to purify not only N. ceranae spores but also other Nosema spp. spores.

Elucidation of Bacillus subtilis KATMIRA 1933 Potential for Spore Production in Submerged Fermentation of Plant Raw Materials.

In this study, the effects of several key factors to increase spore production by Bacillus subtilis subsp. KATMIRA 1933 were evaluated in shake flask experiments. In a synthetic medium, glucose concentration played a crucial role in the expression of bacilli sporulation capacity. In particular, maximum spore yield (2.3×109 spores/mL) was achieved at low glucose concentration (2g/L), and further gradual increase of the carbon source content in the medium caused a decrease in sporulation capacity. Substitution of glucose with several inexpensive lignocellulosic materials was found to be a reasonable way to achieve high cell density and sporulation. Of the materials tested, milled mandarin peels at a concentration of 40g/L served as the best growth substrate. In these conditions, bacilli secreted sufficient levels of glycosyl hydrolases, providing slow hydrolysis of the mandarin peel's polysaccharides to metabolizable sugars, providing the bacterial culture with an adequate carbon and energy source. Among nitrogen sources tested, peptone was found to favor spore production. Moreover, it was shown that cheese and cottage cheese whey usage, instead of distilled water, significantly increases spore formation. After optimization of the nutrient medium in the shake flask experiments, the technical feasibility of large-scale spore production by B. subtilis KATMIRA 1933 was confirmed in a laboratory fermenter. The spore yield (7×1010 spores/mL) obtained using a bioreactor was higher than those previously reported.

Optimization of a Binary Concrete Crack Self-Healing System Containing Bacteria and Oxygen.

An optimized strategy for the enhancement of microbially induced calcium precipitation including spore viability ensurance, nutrient selection and O2 supply was developed. Firstly, an optimal yeast extract concentration of 5 g/L in sporulation medium was determined based on viable spore yield and spore viability. Furthermore, the effects of certain influential factors on microbial calcium precipitation process of H4 in the presence of oxygen releasing tablet (ORT) were evaluated. The results showed that CaO2 is preferable to other peroxides in improving the calcium precipitation by H4. H4 strain is able to precipitate a highly insoluble calcium at the CaO2 dosage range of 7.5–12.5 g/L, and the most suitable spore concentration is 6 × 108 spores/ml when the spore viability (viable spore ratio) is approximately 50%. Lactate is the best carbon source and nitrate is the best nitrogen source for aerobic incubation. This work has laid a foundation of ternary self-healing system containing bacteria, ORT, and nutrients, which will be promising for the self-healing of cracks deep inside the concrete structure.

High concentrations of intracellular Ap4A and/or Ap5A in developing Myxococcus xanthus cells inhibit sporulation

Diadenosine polyphosphates (ApnA) are thought to act as signalling molecules regulating stress responses and biofilm formation in prokaryotes. However, ApnA function in Myxococcus xanthus remains unknown. Here, we investigated the role of ApnA in M. xanthus, using the wild-type and ApnA hydrolase (apaH) mutant strains exposed to various stress conditions. In both wild-type and apaH mutant cells cultured on starvation medium (CF agar), the levels of intracellular diadenosine tetraphosphate (Ap4A) and pentaphosphate (Ap5A) increased several fold during the first 16 h of development and decreased gradually thereafter. The levels of Ap4A and Ap5A in the apaH mutant were about 5- and 11-fold higher than those in the wild-type strain at 16 h, respectively. ApnA hydrolase activity of the wild-type strain increased 1.5-fold during the first 8 h of development, and it then gradually decreased. The apaH mutant formed spores 1-2 days after the wild-type strain did, and the yield of viable spores was 5.5 % of that in the wild-type strain 5 days after inoculation onto CF agar. These results suggest the possibility that high intracellular levels of Ap4A and/or Ap5A may inhibit M. xanthus sporulation at the early stage of development and that the bacteria reduce intracellular Ap4A and Ap5A accumulation through ApnA hydrolase activity.

Functional Analysis of the Pathogenicity-Related Gene VdPR1 in the Vascular Wilt Fungus Verticillium dahliae.

Verticillium dahliae Kleb., the causal agent of vascular wilt, can seriously diminish the yield and quality of many crops, including cotton. The pathogenic mechanism to cotton is complicated and unclear now. To screen pathogencity related genes and identify their function is the reliable way to explain the mechanism. In this study, we obtained a low-pathogenicity mutant vdpr1 from a T-DNA insertional library of the highly virulent isolate of V. dahliae Vd080, isolated from cotton. The tagged gene was named pathogenicity-related gene (VdPR1). The deletion mutant ΔVdPR1 did not form microsclerotia and showed a drastic reduction in spore yield and mycelial growth, compared to wild type. Also, ΔVdPR1 showed significantly lower protease and cellulase activities than those of wild type. Complementation of the mutant strain with VdPR1 (strain ΔVdPR1-C) almost completely rescued the attributes described above to wild-type levels. The knockout mutant ΔVdPR1 showed delayed infection, caused mild disease symptoms, formed a smaller biomass in roots of the host, and showed compromised systemic invasive growth in the xylem. These results suggest that VdPR1 is a multifaceted gene involved in regulating the growth development, early infection and pathogenicity of V. dahliae.

Suppressive subtractive hybridization reveals different gene expression between high and low virulence strains of Cladosporium cladosporioides

Cladosporium cladosporioides is a ubiquitous fungus, causing infections in plants, humans, and animals. Suppression subtractive hybridization (SSH) and quantitative real-time PCR (qRT-PCR) were used in this study to identify differences in gene expression between two C. cladosporioides strains, the highly virulent Z20 strain and the lowly virulent Zt strain. A total of 61 unigenes from the forward library and 42 from the reverse library were identified. Gene ontology (GO) analysis showed that these genes were involved in various biological processes, cellular components and molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the unigenes in the forward library corresponded to 5 different pathways and the reverse library unigenes were involved in 3 different pathways. The qRT-PCR results indicated that expressions of APL1, GUD1, CSE1, SPBC3E7.04c and MFS were significantly different between Z20 and Zt strains, while genes encoding the senescence-associated proteins, pse1, nup107, mip1, pex2, icl1 and α/β hydrolase exhibited no significant differences between the two strains. In addition, we found that 5 unigenes encoding mip1, chk1, icl1, α/β hydrolase and β-glucosidase may be associated with pathogenicity. One unigene (MFS) may be related to the resistance to 14 α-demethylase inhibitor fungicides, and 5 unigenes (PEX2, NUP107, PSE1, APL1, and SPBC3E7.04c) may be related to either low spore yield or earlier aging of the Zt strain. Our study may help better understand the molecular mechanism of C. cladosporioides infection, and therefore improve the treatment and prevention of C. cladosporioides induced diseases.

Screening of Fungal Strains Grown in Solid-state Culture for Production of Pectinase from Coffee Husk

Eighty percent of Vietnamese coffee production can be found in Central Highlands (Tay Nguyen). This paper describes a screening of fungi strains isolated from coffee husk waste collected in Dak Lak province, Tay Nguyen, for pectinase production. It was found that 17 different fungi strains were isolated from samples of 11 coffee farms. Among them. there were only 9 trains which could hydrolyze pectin. The diameter of the hydrolysis halo around fungi colonies in Pectinase Screening Agar Medium (PSAM) was measured as an indicator to assess the pectinase activity. Phylogenetic analysis based on 28S rRNA gene sequences showed that detected Rhizopus oryzae . Aspergillus oryzae and Hypocrea pseudokoningii were those giving the largest holo zones. Hypocrea pseudokoningii presented the best pectinase activity of 657.16 UI/g and was chosen for biomass production to collect enzyme. In a further study, effect of rice bran addition to coffee husk and moisture of culture medium on the spore yield of Hypocrea pseudokoningii were investigated. Using coffee husk medium with 23% rice bran addition and 65% moisture at ambient temperature, the highest spore yield of 9.2 x10 8 spores/g was found after incubation for 168 hours. The fungi biomass product was dried at 40 o C for 54 hours to obtain the final moisture of 12% and spore survival of 5.9 x10 8 spores/g.