An image analysis technique to estimate the cell density and biomass concentration of Trichoderma reesei

Aim:  The objective is to develop an automated image analysis protocol to quantify the cell volume fraction of filamentous fungi ( Trichoderma reesei ) and estimate the biomass concentration.
Methods and Results:  Both dry weight and image analyses were performed on samples collected periodically from 7-l stirred tank fermentations. Using the projected area of lactophenol blue-stained hyphae, the fraction occupied by the cells in a given volume was estimated. Combined with the biomass dry weight obtained by filtration, the method was used to estimate the density of filamentous fungi. Knowing the density of fungi, the algorithm was employed to quantitatively assess the biomass evolution during the course of fermentation even in the presence of solid particles.
Conclusions:  A density of 0.334 g dry weight cm⁻³ was found for T. reesei RUT C-30. The image analysis protocol allowed successful estimation of biomass concentration in the presence or absence of solid particles.
Significance and Impact of the Study:  Methods to quantify biomass during the industrial production of cellulase with T. reesei are often limited due to the presence of solid substrates. The image analysis protocol presented here offers a quick and easy way to estimate biomass concentration of filamentous micro-organisms in insoluble medium.     

Growth of acetotrophic,methane-producing bacteria in a pH auxostat

Three acetotrophicMethanosarcina species, which included marine, nonmarine, and thermophilic strains, were grown on acetate in a 10-liter pH auxostat. Specific growth rates and molar growth yields were constant throughout growth. Cell yields were up to 18-fold greater than previously reported. These properties of the pH auxostat indicate that it is a preferred culture method for the biochemical study of methanogenesis from acetate.     

Ozone as a selective disinfectant for nonaseptic fungal cultivation on corn-processing wastewater

Treatment of wet corn-milling wastewater with filamentous fungi was investigated as a means of obtaining fungal biomass as an additional byproduct. Competitive bacterial growth is a common problem during this nonaseptic treatment process. Selective disinfection with ozone was evaluated for eliminating bacterial populations during fungal cultivation. Three laboratory-scale continuous flow aerated reactors were operated under nonaseptic conditions at 38 degrees C, hydraulic retention time of 8h and pH of 4. The bacterial population was reduced by one log with respect to the control when ozone was dosed at a concentration above 47+/-2mg/L. An ozone dosage of about 57mg/L was found to be most effective in improving both fungal biomass production and soluble chemical oxygen demand (SCOD) removal (up to 90%). Fungal biomass concentration increased from c. 1.45g/L (control) to c. 1.75g/L at a 57-mg/L ozone dosage. Higher and lower dosages of ozone resulted in poorer fungal growth and lower SCOD removal.     

Alkane utilisation by Cladosporium resinae: the importance of extended lag phases when assessing substrate optima

Abstract Lag phases were shown to be important when assessing substrate optima for alkane utilisation by filamentous fungi. Failure to take the progressively longer lag phase (in respect to carbon chain length) into consideration may have led previous workers, who used single point biomass measurements as an indication of growth, to underestimate the potential of fungi to grow on alkanes such as octadecane. For a variety of fungi tested, optimum substrates of somewhat longer chain-length than normally reported were found by using growth rates as indicators of best growth, e.g., octadecane for Cladosporium resinae .     

Removal of thorium from simulated acid process streams by fungal biomass

Biomass from several fungal species removed thorium from solution in 1M HNO(3), pH 0-1. Thorium uptake was saturable with increasing thorium concentration, although the equilibria did not correspond to a simple ad sorption isotherm. Thorium uptake was altered by the biomass concentration, the uptake per unit biomass being reduced at high biomass concentrations. The presence of Al(3+) and Fe(3+) only slightly inhibited uptake of thorium while Ca(2+), Mg(2+), and Na(+) had no effect. Thus fungal biomass appears capable of removing thorium from solution under chemical conditions existing in acid waste liquors. Thorium uptake was increased by pretreatment using detergent and also, in the case of filamentous fungi, varied with the culture conditions, which implies that the thorium uptake characteristics of fungal biomass are able to be manipulated by these or similar means for optimum performance.     

Fungal biodegradation of dibutyl phthalate and toxicity of its breakdown products on the basis of fungal and bacterial growth

Phthalates are esters of phthalic acid that give flexibility to polyvinyl chloride. Diverse studies have reported that these compounds might be carcinogenic, mutagenic and/or teratogenic. Radial growth rate, biomass, hyphal thickness of Neurospora sitophyla, Trichoderma harzianum and Aspergillus niger, grown in two different concentrations of dibutyl phthalate (DBP) (500 and 1,000 mg/l) in agar and in submerged fermentation were studied. The inhibitory concentration (IC₅₀) and the constant of biodegradation of dibutyl phthalate in Escherichia coli cultures were used to evaluate toxicity. The radial growth rate and thickness of the hypha were positively correlated with the concentration of phthalate. The pH of the cultures decreased as the fermentation proceeded. It is shown that these fungi are able to degrade DBP to non-toxic compounds and that these can be used as sole carbon and energy sources by this bacterium. It is demonstrated that the biodegradation of the DBP is directly correlated with the IC₅₀. This is the first study that reports a method to determine the biodegradation of DBP on the basis of the IC₅₀ and fungal growth, and the effect of this phthalate on the growth and thickness of hyphae of filamentous fungi in agar and in submerged fermentation.     

Trichoderma: the genomics of opportunistic success

Trichoderma is a genus of common filamentous fungi that display a remarkable range of lifestyles and interactions with other fungi, animals and plants. Because of their ability to antagonize plant-pathogenic fungi and to stimulate plant growth and defence responses, some Trichoderma strains are used for biological control of plant diseases. In this Review, we discuss recent advances in molecular ecology and genomics which indicate that the interactions of Trichoderma spp. with animals and plants may have evolved as a result of saprotrophy on fungal biomass (mycotrophy) and various forms of parasitism on other fungi (mycoparasitism), combined with broad environmental opportunism.     

Breakdown of plant cell biomass by filamentous fungi

Three species of filamentous fungi, Botrytis cinerea, Sporotrichum thermophile and Trichoderma viride, have been selected to assess the potential of utilizing filamentous fungi to degrade plant cell biomass produced by mass cell culture techniques. All three fungal species grew comparatively well on plant cell biomass with no requirement for supplementary nutrients. Of the three species assessed B. cinerea demonstrated the most growth. This species also produced the greatest yield of d-glucose. However, when culture conditions were modified, yields of d-glucose were markedly reduced indicating that the combination of species and culture conditions must be thoroughly investigated to ensure maximum product yield. The growth of filamentous fungi on plant cells also markedly affected the nature of the resulting fungal-plant cell residue, increasing the levels of soluble carbohydrates and essential amino acids with the largest increase in these materials being promoted by B. cinerea.     

Factors affecting population development within seawater-displaced fuel tanks

Abstract Populations within model seawater-displaced fuel tanks developed as a succession of eukaryotic species. Growth of filamentous fungi was preceded by the growth of several yeast species. More rapid initiation of growth by filamentous fungi was associated with extracellular metabolites produced by yeasts: both the pH flux towards more acidic conditions and the spore germination induction effect influenced population development. The relative binding affinity of each species for the insoluble hydrocarbon fuel is proposed as a major characteristic determining which species proliferated at the fuel-water interface.     

Hexavalent chromium removal in vitro and from industrial wastes, using chromate-resistant strains of filamentous fungi indigenous to contaminated wastes

Two chromate-resistant filamentous fungi, strains H13 and Ed8, were selected from seven independent fungal isolates indigenous to Cr(VI)-contaminated soil because of their ability to decrease hexavalent chromium levels in the growth medium. Morphophysiological studies identified strain H13 as a Penicillium sp. isolate and Ed8 as an Aspergillus sp. isolate. When incubated in minimal medium with glucose as a carbon source and in the presence of 50 microg/mL Cr(VI), these strains caused complete disappearance of Cr(VI) in the growth medium after about 72 h of incubation. Total chromium concentration in growth medium was constant during culture growth, and no accumulation of chromium in fungal biomass was observed. Quantitative determinations of oxidized and reduced chromium species during the reduction process revealed stoichiometric conversion of Cr(VI) to Cr(III). A decrease in Cr(VI) levels from industrial wastes was also induced by Ed8 or H13 biomass. These results indicate that chromate-resistant filamentous fungi with Cr(VI)-reducing capability could be useful for the removal of Cr(VI) contamination.     

The ability of filamentous fungi to produce acids on indoor building materials

Sixty two filamentous fungi isolated from paint coatings, wallpaper, carton-gypsum board, and indoor air in buildings were screened for acid activity. It was found that 64.5% of strains produce acids on medium with bromo-cresol purple, where 18% of the strains were distinguished by very high acid activity (acid activity coefficient Q = 1.32–2.83), including the species:Aspergillus niger, Aspergillus versicolor, Penicillium expansum, Penicillium brevicom pactum, Penicillium chrysogenum, Cladosporium cladosporioides, Stachybotrys chartarum, Mucor globosus, Ulocladium chartarum andAlternaria alternata. Research indicated that filamentous fungi considerably decrease the pH of the medium when that medium containing building material. The greatest acid production and pH decrease of the medium was observed during the growth of filamentous fungi in a medium with mortar, while the production of acids was less in a medium with cartongypsum board, gypsum, and wallpaper. Filamentous fungi produced succinic, oxalic, malic and fumaric acids in the medium with indoor building materials. It was stated that the type of building material affects the spectrum and quantity of organic acids produced by filamentous fungi.     

Influence of ultrasound amplitude and duty cycle on fungal morphology and broth rheology of <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

Effects of ultrasound amplitude and duty cycle on cultures of Aspergillus terreus are reported in a 25 l slurry bubble column sonobioreactor. Fermentations were carried out batchwise. A 2^k-factorial design with added central points was used. Sonication at any cycle and amplitude level did not affect biomass growth rate and yield relative to nonsonicated control, but did affect growth morphology. Ultrasound disrupted fungal pellets and caused the biomass to grow mainly as dispersed hyphae. Production of lovastatin was reduced by medium- and high-cycle sonication. Sonication affected broth rheology. In view of these results, sonication can be used to modify growth morphology and broth rheology without affecting growth rate and yield of filamentous fungi.     

Feasibility of filamentous fungi for biofuel production using hydrolysate from dilute sulfuric acid pretreatment of wheat straw

ABSTRACT: BACKGROUND: Lipids produced from filamentous fungi show great promise for biofuel production, but a major limiting factor is the high production cost attributed to feedstock. Lignocellulosic biomass is a suitable feedstock for biofuel production due to its abundance and low value. However, very limited study has been performed on lipid production by culturing oleaginous fungi with lignocellulosic materials. Thus, identification of filamentous fungal strains capable of utilizing lignocellulosic hydrolysates for lipid accumulation is critical to improve the process and reduce the production cost. RESULTS: The growth performances of eleven filamentous fungi were investigated when cultured on glucose and xylose. Their dry cell weights, lipid contents and fatty acid profiles were determined. Six fungal strains with high lipid contents were selected to culture with the hydrolysate from dilute sulfuric acid pretreatment of wheat straw. The results showed that all the selected fungal strains were able to grow on both detoxified liquid hydrolysate (DLH) and non-detoxified liquid hydrolysate (NDLH). The highest lipid content of 39.4% was obtained by Mortierella isabellina on NDLH. In addition, NDLH with some precipitate could help M. isabellina form pellets with an average diameter of 0.11 mm. CONCLUSION: This study demonstrated the possibility of fungal lipid production from lignocellulosic biomass. M. isabellina was the best lipid producer grown on lignocellulosic hydrolysates among the tested filamentous fungi, because it could not only accumulate oils with a high content by directly utilizing NDLH to simplify the fermentation process, but also form proper pellets to benefit the downstream harvesting. Considering the yield and cost, fungal lipids from lignocellulosic biomass are promising alternative sources for biodiesel production.     

一种便于丝状真菌显微观察的培养方法

在真菌培养过程中,对其个体形态与群体（菌落）形态进行实时观察与鉴定是很必要的。本文利用半培养基培养法结合显微操作技术,对丝状真菌个体与群体进行形态学观察。结果表明,该方法无需染色与制片,不破坏菌丝正常生长状态,可实时进行形态学检测,对多个菌种在自然生长状态下的菌丝与菌落特征进行观察,操作简单、方便、快捷,从而降低了成本及工作量。     

Lead biosorption by different morphologies of fungus Mucor indicus

Biosorption characteristics of Pb⁺² ions from aqueous solution were investigated using fungus Mucor indicus biomass treated with NaOH. Biosorption was measured as a function of biomass morphology, pH, biomass concentration, contact time, and metal concentration. The morphology of M. indicus biomass was manipulated towards filamentous or yeast-like forms. The highest and lowest biosorption capacities were observed for purely filamentous and yeast-like forms, respectively. Models of Langmuir, Freundlich, Temkin, and Scachard were applied to describe adsorption isotherm and fitted appropriately. Biosorption kinetics was successfully described using Ho’s pseudo-second-order model. Maximum and minimum values of biosorption capacity of Pb²⁺ were 22.1 and 12.1 mg g⁻¹ for purely filamentous and yeast-like morphologies, respectively. Increasing pH resulted in higher biosorption of Pb⁺² ions up to pH 5.5. Biosorption capacity of individual Pb⁺² ions was reduced in the presence of other metal ions in bi- or multi-metal ion experiments. Metal ions adsorption by the biomass could be eluted effectively with HNO₃.     

Application of monoclonal antibodies in quantifying fungal growth dynamics during aerobic spoilage of silage

Proliferation of filamentous fungi following ingress of oxygen to silage is an important cause of dry matter losses, resulting in significant waste. In addition, the production of mycotoxins by some filamentous fungi poses a risk to animal health through mycotoxicosis. Quantitative assessment of fungal growth in silage, through measurement of ergosterol content, colony-forming units or temperature increase is limiting in representing fungal growth dynamics during aerobic spoilage due to being deficient in either representing fungal biomass or being able to identify specific genera. Here, we conducted a controlled environment aerobic exposure experiment to test the efficacy of a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) to detect the proliferation of fungal biomass in six silage samples. We compared this to temperature which has been traditionally deployed in such experiments and on-farm to detect aerobic deterioration. In addition, we quantified ergosterol, a second marker of fungal biomass. After 8 days post-aerobic exposure, the ergosterol and ELISA methods indicated an increase in fungal biomass in one of the samples with a temperature increase observed after 16 days. A comparison of the methods with Pearson's correlation coefficient showed a positive association between temperature and ergosterol and both markers of fungal biomass. This work indicates that the technology has potential to be used as an indicator of microbial degradation in preserved forage. Consequently, if it developed as an on-farm technique, this could inform forage management decisions made by farmers, with the goal of decreasing dry matter losses, improving resource and nutrient efficiency and reducing risks to animal health.     

丝状真菌纤维素酶合成诱导及转录调控

利用廉价可再生木质纤维素资源水解产生的可发酵糖生产生物能源和生物基化学品是近年来国内外研究的热点。纤维素酶酶解是木质纤维素原料生物降解的重要手段,但目前纤维素酶生产成本过高,限制了纤维素生物转化和生物炼制的工业化应用。对丝状真菌纤维素酶基因表达和调控进行研究,有利于进一步选育纤维素酶高产菌株,降低纤维素酶生产成本。随着高通量测序及丝状真菌遗传操作等技术的进步,对丝状真菌纤维素酶诱导和基因表达调控机理有了更深入的认识。本文综述了近年来丝状真菌纤维素酶诱导和纤维素酶基因表达调控的最新进展,重点论述糖转运蛋白、转录因子和染色质重塑对纤维素酶表达调控的影响,并对利用人工锌指蛋白进行丝状真菌纤维素酶诱导调控研究进行了展望。     

Rapid microbial growth in a pH auxostat

Summary Feeding nutrient to meet demand dilutes slow-growing organisms from continuous culture and greatly favors rapid growth. Doubling times of roughly 10 minutes have been verified in a pH auxostat by viable cell counts and by direct counting with a Petroff-Hausser chamber. Effects of wall attachment were negligible because a fresh reactor was substituted frequently before wall growth could become established.     

接种彩色豆马勃对模拟酸沉降下马尾松幼苗生物量的影响

外生菌根能够提高宿主植物对外界环境胁迫的抵抗力,促进植物的生长,本文试图揭示外生菌根对酸雨胁迫下马尾松生长的保护作用。本研究采用盆栽试验,共设置四个处理：酸雨对照处理（Control check (CK), 约pH值5.5）不接种,酸雨对照处理接种,酸雨pH值3.5处理不接种,酸雨pH值3.5处理接种。pH值3.5的酸雨处理降低马尾松的生物量,在试验前期降低根冠比,试验中后期则提高根冠比,在试验初期增加了叶面积,但中后期显著降低了叶面积。接种外生菌根菌有利于马尾松幼苗的生长,pH值3.5处理下接种外生菌根菌能提高马尾松幼苗的生物量,外生菌根菌对生物量分配和叶面积的影响与酸雨胁迫的影响是相反的,即外生菌根菌抵消了酸雨胁迫对马尾松的影响。     

Fungal and bacterial growth responses to N fertilization and pH in the 150-year 'Park Grass' UK grassland experiment

The effects of nitrogen (N) fertilization (0-150 kg N ha?1 year?1 since 1865) and pH (3.3-7.4) on fungal and bacterial growth, biomass and phospholipid fatty acid (PLFA) composition were investigated in grassland soils from the 'Park Grass Experiment', Rothamsted Research, UK. Bacterial growth decreased and fungal growth increased with lower pH, resulting in a 50-fold increase in the relative importance of fungi between pH 7.4 and 3.3. The PLFA-based fungal:bacterial biomass ratio was unchanged between pH 4.5 and 7.4, and decreased only below pH 4.5. Respiration and substrate-induced respiration biomass both decreased three- to fourfold with lower pH, but biomass concentrations estimated using PLFAs were unaffected by pH. N fertilization did not affect bacterial growth and marginally affected fungal growth while PLFA biomass marker concentrations were all reduced by higher N additions. Respiration decreased with higher N application, suggesting a reduced quality of the soil organic carbon. The PLFA composition was strongly affected by both pH and N. A comparison with a pH gradient in arable soil allowed us to generalize the pH effect between systems. There are 30-50-fold increases in the relative importance of fungi between high (7.4-8.3) and low (3.3-4.5) pH with concomitant reductions of respiration by 30-70%.