Zhiming Zheng

Growth and metabolism of Beauveria bassiana spores and mycelia

BackgroundFungi are ubiquitous in nature and have evolved over time to colonize a wide range of ecosystems including pest control. To date, most research has focused on the hypocrealean genera Beauveria bassiana, which is a typical filamentous fungus with a high potential for insect control. The morphology and components of fungi are important during the spores germination and outgrow to mycelia. However, to the best of our knowledge, there is no report on the morphology and components of B. bassiana spores and mycelia. In the work, the growth and metabolism of Beauveria bassiana spores and mycelia were studied. High performance liquid chromatography-mass spectrometry (HPLC-MS) was employed to study the metabolism of B. bassiana spores and mycelia. Principal component analysis (PCA) based on HPLC-MS was conducted to study the different components of the spores and mycelia of the fungus. Metabolic network was established based on HPLC-MS and KEGG database.ResultsThrough Gompertz model based on macroscopic and microscopic techniques, spore elongation length was found to increase exponentially until approximately 23.1xa0h after cultivation, and then growth became linear. In the metabolic network, the decrease of glyoxylate, pyruvate, fumarate, alanine, succinate, oxaloacetate, dihydrothymine, ribulose, acetylcarnitine, fructose-1, 6-bisphosphate, mycosporin glutamicol, and the increase of betaine, carnitine, ergothioneine, sphingosine, dimethyl guanosine, glycerophospholipids, and in spores indicated that the change of the metabolin can keep spores in inactive conditions, protect spores against harmful effects and survive longer.ConclusionsAnalysis of the metabolic pathway in which these components participate can reveal the metabolic difference between spores and mycelia, which provide the tools for understand and control the process of of spores germination and outgrow to mycelia.

Electromagnetic Attenuation Characteristics of Microbial Materials in the Infrared Band

In this study, seven microbial materials (entomogenous fungi Bb3088 mycelia, entomogenous fungi Bb3088 spores, entomogenous fungi Ma2677 mycelia, entomogenous fungi Ma2677 spores, Bacillus subtilis 8204, Staphylococcus aureus 6725, and Saccharomyces cerevisiae 1025) were used to measure electromagnetic (EM) signal extinction. They were subjected to light absorption and reflection measurements in the range of 4000–400u2009cm−1 (2.5–25u2009µm) using Fourier transform infrared spectroscopy. The specular reflection spectrum method was used to calculate the real (n) and imaginary (k) parts of the complex refractive index. The complex refractive index with real part n and imaginary part k in the infrared band satisfies the following conditions nu2009≥u20091 and ku2009≥u20090. The mass extinction coefficient was calculated based on Mie theory. Entomogenous fungi Ma2677 spores and entomogenous fungi Bb3088 spores were selected as EM signal extinction materials in the smoke box test. The transmittances of entomogenous fungi Bb3088 spores and entomogenous fungi Ma2677 spores were 11.63% and 5.42%, and the mass extinction coefficients were 1.8337u2009m2/g and 1.227u2009m2/g. These results showed that entomogenous fungi Bb3088 spores and entomogenous fungi Ma2677 spores have higher extinction characteristics than other microbial materials.

Optimised fermentation conditions and improved collection efficiency using dual cyclone equipment to enhance fungal conidia production

Methods for enhancing conidial yield and for harvesting pure fungal conidia of entomopathogenic fungi were investigated. Fermentation conditions (liquid-to-solid ratio, MgSO4·7H2O, incubation temperature, inoculum sizes, KNO3 and relative humidity) of Beauveria bassiana s.l. and Metarhizium anisopliae s.l. were optimised to increase the conidial yields that reached 11.2 mg/g and 24.5 mg/g, increases of 72% and 52% compared to the unoptimised yields of 6.5 mg/g and 16.1 mg/g, respectively. Three methods were compared for harvesting pure conidia of B. bassiana: dual cyclone equipment (DCE), sieving 200 and elution with 0.02% Tween-80 suspension. DCE performed the best, giving a conidial yield of 12.6 mg/g and 1.8 × 1010 conidia·g–1. To further enhance the harvest efficiency, response surface methodology combined with a Box–Behnken design was employed, and the conidial yield of B. bassiana reached 20.9 mg/g, a total increase of 221% compared to the original conditions. Under these optimised harvest parameters, the conidial yield of M. anisopliae rose to 42.2 mg/g, an increase of 162%. The conidia of B. bassiana and M. anisopliae harvested in this way were pure, with no mycelial fragments or substrate visible in microscopic images.

Hydrodynamic investigation of a novel shear-generating device for the measurement of anchorage-dependent cell adhesion intensity

Proliferation of anchorage-dependent cells occurs after adhesion to a suitable surface. Thus, quantitative information about the force of cells adhesion to microcarriers at early culture phases is vital for scaling up such system. In this work, a newly designed shear-generating device was proposed, based on a previously proposed contraction flow device designed for suspended cells. A design equation for the new device was also proposed to correlate the generated energy dissipation rate (EDR) with the cross-sectional area and flow rate. Microscopic-particle image velocimetry was measured to validate the simulation results, and good agreement was achieved. The newly designed device was then used to measure the adhesion force of MDCK and PK cells, and the results showed that the critical EDR was 3000xa0W/m3 for MDCK and 5000xa0W/m3 for PK cells. This quantitative information is of great value for better understanding shearing effects during the scaling up of anchorage-dependent cell cultures.

Infrared Extinction Performance of Randomly Oriented Microbial-Clustered Agglomerate Materials

In this study, the spatial structure of randomly distributed clusters of fungi An0429 spores was simulated using a cluster aggregation (CCA) model, and the single scattering parameters of fungi An0429 spores were calculated using the discrete dipole approximation (DDA) method. The transmittance of 10.6u2009µm infrared (IR) light in the aggregated fungi An0429 spores swarm is simulated by using the Monte Carlo method. Several parameters that affect the transmittance of 10.6u2009µm IR light, such as the number and radius of original fungi An0429 spores, porosity of aggregated fungi An0429 spores, and density of aggregated fungi An0429 spores of the formation aerosol area were discussed. Finally, the transmittances of microbial materials with different qualities were measured in the dynamic test platform. The simulation results showed that the parameters analyzed were closely connected with the extinction performance of fungi An0429 spores. By controlling the value of the influencing factors, the transmittance could be lower than a certain threshold to meet the requirement of attenuation in application. In addition, the experimental results showed that the Monte Carlo method could well reflect the attenuation law of IR light in fungi An0429 spore agglomerates swarms.

Discrimination of viable and dead microbial materials with Fourier transform infrared spectroscopy in 3–5 micrometers

We present a method to show that average mass extinction coefficient of microbes evaluated via Lorenz-Mie theory can be used to discriminate between viable and dead microbes. Reflectance of viable and dead self-cultured fungal spores and mycelia were measured by the Fourier transform infrared spectroscopy. Complex refractive indices and mass extinction coefficient of viable and dead fungal spores and mycelia were obtained in terms of Kramers-Kronig (KK) relation and Lorenz-Mie theory respectively. Smoke box experimental system was built to validate the effectiveness of the method. The results show that viable and dead fungal spores and mycelia via average mass extinction coefficients can be distinguished. The method can be used to discriminate the bioactivity of microbes and has potential applications in identification, detection, and optical characteristics of viable and dead microbial materials.

Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing

The mycelial morphology of Aspergillus niger, a major filamentous fungus used for citric acid production, is important for citric acid synthesis during submerged fermentation. To investigate the involvement of the chitin synthase gene, chsC, in morphogenesis and citric acid production in A. niger, an RNAi system was constructed to silence chsC and the morphological mutants were screened after transformation. The compactness of the mycelial pellets was obviously reduced in the morphological mutants, with lower proportion of dispersed mycelia. These morphological changes have caused a decrease in viscosity and subsequent improvement in oxygen and mass transfer efficiency, which may be conducive for citric acid accumulation. All the transformants exhibited improvements in citric acid production; in particular, chsC-3 showed 42.6% higher production than the original strain in the shake flask. Moreover, the high-yield strain chsC-3 exhibited excellent citric acid production potential in the scale-up process.The citric acid yield and the conversion rate of glucose of chsC-3 were both improved by 3.6%, when compared with that of the original strain in the stirred tank bioreactor.