Biomass estimation in solid state fermentation II. On-line measurements

Summary Two methods for on-line biomass measurement were tested with success: (a) infrared (IR) estimation of cell components (glucosamine and ergosterol) and medium residues (sucrose and nitrogen) directly on solid medium; (b) the CO₂ evolution rate during cultivation. These methods were very satisfactory for following biomass changes during a defined process. The IR measurements correlated well with manual methods. The correlation between CO₂ and glucosamine measurements was very satisfactory with good precision. However, they did not permit comparison between processes.Offprint requests to: C. Desgranges     

The extraction and quantification of ergosterol from ectomycorrhizal fungi and roots

Recently, ergosterol analysis has been used to quantify viable fungal biomass in resynthesized ectomycorrhizae. An objective of our study was to quantify ergosterol in a range of ectomycorrhizal isolates under differing growth conditions. In addition, we tested the applicability of the method on field-collected roots of ectomycorrhizal and vesicular-arbuscular (VA) mycorrhizal plants. Quantification of sitosterol as a biomass indicator of plant roots was also undertaken. Ergosterol was not detected in roots of uninoculated Betula populifolia seedlings, and sitosterol was not detected in an ectomycorrhizal fungal isolate but was present in birch roots. Ergosterol was produced in all isolates examined, which represented the major orders of ectomycorrhizal fungi. The range of values obtained, from 3 to nearly 18 g ergosterol mg^-1 dry mass, agrees well with reported values for other mycorrhizal and decomposer fungi. Hyphal ergosterol was the same during growth on phytic acid and KH₂PO₄. Reduction of growth temperature from 25° C to 15° C had little effect on ergosterol content of cultures harvested at similar growth stages. Ergosterol and sitosterol were detected in field-collected ectomycorrhizae of B. populifolia and Pinus sylvestris and VA mycorrhizae of Acer rubrum and Plantago major. Both ergosterol content and ergosterol to sitosterol ratios were significantly lower in VA mycorrhizae than ectomycorrhizae. Calculations of viable fungal biomass associated with field-collected roots were in agreement with those reported by others using the method on resynthesized ectomycorrhizae. Estimates of total mass could be obtained for field-collected B. populifolia roots by a simultaneously using ergosterol to estimate fungal biomass and sitosterol to estimate root mass. Some potential applications and limitations of sterol quantification in studies of mycorrhizal physiology and ecology are discussed.     

Effect of culture conditions on the biomass determination by ergosterol of <Emphasis Type="Italic">Lentinus crinitus</Emphasis> and <Emphasis Type="Italic">Psilocybe castanella</Emphasis>

Estimating fungal growth is important in processes of soil bioremediation. It has been demonstrated that ergosterol is a good indicator of fungal biomass in solid substrata. In the present study were evaluated the effects upon the ergosterol rate of Lentinus crinitus Berk. and Psilocybe castanella Peck through the culture conditions of these fungi, which are evaluated for the bioremediation of soils contaminated by organochlorates. A good correlation between fungal biomass and ergosterol was observed for both species. The culture conditions did not influence the ergosterol rate of L. crinitus. Yet the ergosterol rate of P. castanella was influenced from 35 days of culture and when grown in the presence of 15.00 g hexachlorobenzene l⁻¹ of culture medium. So it is possible to estimate growth of both species using ergosterol as indicator in processes of soil bioremediation since the influences observed in the ergosterol rate of P. castanella are considered.     

Effect of pollutants on the ergosterol content as indicator of fungal biomass

Ergosterol content was determined in 20 white-rot fungi isolates and the values ranged from 2380 to 13 060 μg g⁻¹ fungal biomass. Significant changes of ergosterol content according the physiological stage for Bjerkandera adusta 4312 and Coriolopsis gallica 8260 were found, showing the highest values during the stationary phase. However, in the case of Phanerochaete chrysosporium 3642, no changes were detected during growth. The effect of pollutants, such as heavy metals and fungicides, on the ergosterol content of C. gallica was determined. Heavy metals (Cu 80 ppm, Zn 50 ppm or Cd 10 ppm) and fungicides (thiram 3 ppm or pentachlorophenol 1.5 ppm) at concentrations that reduce the metabolic activity between 18% and 53% (pollutant-stressed cultures) did not affect the ergosterol content. Only the fungicide zineb (25 ppm) reduced significantly the ergosterol content in biomass basis. In soil experiments with Cu (80 ppm) or thiram (10 ppm) after 15 and 30 days of incubation, the ergosterol content in soil was linearly correlated to the fungal biomass C in both polluted and control soil cultures. The ergosterol content was independent of the presence or the absence of pollutants. Thus, these results indicate that ergosterol can be a useful indicator for fungal biomass in polluted soils, and can be applied for monitoring bioremediation processes.     

Changes in Soil Microbial Biomass and Residual Indices as Ecological Indicators of Land Use Change in Temperate Permanent Grassland

The relationship between microbial biomass, residues and their contribution to microbial turnover is important to understand ecosystem C storage. The effects of permanent grassland (100 % ryegrass—PG), conversion to modified grassland (mixture of grass and clover—MG) or maize monoculture (MM) on the dynamics of soil organic C (SOC), microbial biomass, fungal ergosterol and microbial residues (bacterial muramic acid and fungal glucosamine) were investigated. Cattle slurry was applied to quantify the effects of fertilisation on microbial residues and functional diversity of microbial community across land use types. Slurry application significantly increased the stocks of microbial biomass C and S and especially led to a shift in microbial residues towards bacterial tissue. The MM treatment decreased the stocks of SOC, microbial biomass C, N and S and microbial residues compared with the PG and MG treatments at 0–40 cm depth. The MM treatment led to a greater accumulation of saprotrophic fungi, as indicated by the higher ergosterol-to-microbial biomass C ratio and lower microbial biomass C/S ratio compared with the grassland treatments. The absence of a white clover population in the PG treatment caused a greater accumulation of fungal residues (presumably arbuscular mycorrhizal fungi (AMF), which do not contain ergosterol but glucosamine), as indicated by the significantly higher fungal C-to-bacterial C ratio and lower ergosterol-to-microbial biomass C ratio compared with the MG treatment. In addition to these microbial biomass and residual indices, the community level physiological profiles (CLPP) demonstrated distinct differences between the PG and MG treatments, suggesting the potential of these measurements to act as an integrative indicator of soil functioning.     

Fine root chemistry and decomposition in model communities of north-temperate tree species show little response to elevated atmospheric CO<Subscript>2</Subscript> and varying soil resource availability

Rising atmospheric [CO₂] has the potential to alter soil carbon (C) cycling by increasing the content of recalcitrant constituents in plant litter, thereby decreasing rates of decomposition. Because fine root turnover constitutes a large fraction of annual NPP, changes in fine root decomposition are especially important. These responses will likely be affected by soil resource availability and the life history characteristics of the dominant tree species. We evaluated the effects of elevated atmospheric [CO₂] and soil resource availability on the production and chemistry, mycorrhizal colonization, and decomposition of fine roots in an early- and late-successional tree species that are economically and ecologically important in north temperate forests. Open-top chambers were used to expose young trembling aspen (Populus tremuloides) and sugar maple (Acer saccharum) trees to ambient (36 Pa) and elevated (56 Pa) atmospheric CO₂. Soil resource availability was composed of two treatments that bracketed the range found in the Upper Lake States, USA. After 2.5 years of growth, sugar maple had greater fine root standing crop due to relatively greater allocation to fine roots (30% of total root biomass) relative to aspen (7% total root biomass). Relative to the low soil resources treatment, aspen fine root biomass increased 76% with increased soil resource availability, but only under elevated [CO₂]. Sugar maple fine root biomass increased 26% with increased soil resource availability (relative to the low soil resources treatment), and showed little response to elevated [CO₂]. Concentrations of N and soluble phenolics, and C/N ratio in roots were similar for the two species, but aspen had slightly higher lignin and lower condensed tannins contents compared to sugar maple. As predicted by source-sink models of carbon allocation, pooled constituents (C/N ratio, soluble phenolics) increased in response to increased relative carbon availability (elevated [CO₂]/low soil resource availability), however, biosynthetically distinct compounds (lignin, starch, condensed tannins) did not always respond as predicted. We found that mycorrhizal colonization of fine roots was not strongly affected by atmospheric [CO₂] or soil resource availability, as indicated by root ergosterol contents. Overall, absolute changes in root chemical composition in response to increases in C and soil resource availability were small and had no effect on soil fungal biomass or specific rates of fine root decomposition. We conclude that root contributions to soil carbon cycling will mainly be influenced by fine root production and turnover responses to rising atmospheric [CO₂], rather than changes in substrate chemistry.     

Measurement of ergosterol,diaminopimelic acid and glucosamine in soil: evaluation as indicators of microbial biomass

Methods were developed for the measurement of ergosterol, diaminopimelic acid (DAP) and glucosamine in soil as possible indicators of, respectively, fungal, bacterial and total microbial biomass. Ergosterol, obtained by saponification of methanol extracts of soil, was measured by high pressure liquid chromatography with ultra-violet detection. DAP and glucosamine in acid hydrolysates of soil were separated and assayed by quantitative paper chromatography. Physical losses in extraction (generally < 15%) were quantified using ¹⁴C-labelled compounds. Amount (with coefficients of variation) in grassland and arable soils were 0.99–2.06 μg ergosterol (2–16%), 17–163 μg DAP (10–36%) and 505–2109 μg glucosamine (6–23%) per g soil. Evaluation of the DAP and glucosamine figures on the basis of known soil biomass data indicated that these compounds were largely associated with non-living organic matter. In contrast, the ergosterol measured was of the order expected from the fungal biomass present, and this substance may therefore provide a valuable biomass indicator.     

Mathematical models of mycelium growth and ergosterol synthesis in stationary mould culture

Aims:  To develop mathematical models for mycelium growth and ergosterol formation in conditions of periodic stationary culture; to verify possibilities of applying a model describing the relationship between ergosterol content and mycelium quantity.
Methods and Results:  The mould growth and ergosterol formation models covering all phases of mould growth are described using a modified logistic equation with the addition of an exponential function. The correlation between ergosterol and mycelium biomass depended on the growth phase of mould. Meaningful relation was obtained for initial two phases, when both parameters were growing equally. The quadratic function for estimation of the biomass based on ergosterol content was formulated. The error resulting from the application of this function in initial phases of moulds growth was small at 5–7%, in the following phases it was at 11–31%.
Conclusions:  Mycelium biomass can be precisely determined basing on the ergosterol content, when we know the moulds growth phase. In natural environments, without the information about growth phases, it will be possible, but with the higher error.
Significance and Impact of the Study:  Presented model based on the ergosterol content making possible to estimate the quantity of mycelium in moulds cultures and natural environments.     

Magnesium deficiency in sugar beets alters sugar partitioning and phloem loading in young mature leaves

Magnesium deficiency has been reported to affect plant growth and biomass partitioning between root and shoot. The present work aims to identify how Mg deficiency alters carbon partitioning in sugar beet (Beta vulgaris L.) plants. Fresh biomass, Mg and sugar contents were followed in diverse organs over 20 days under Mg-sufficient and Mg-deficient conditions. At the end of the treatment, the aerial biomass, but not the root biomass, of Mg-deficient plants was lower compared to control plants. A clear inverse relationship between Mg and sugar contents in leaves was found. Mg deficiency promoted a marked increase in sucrose and starch accumulation in the uppermost expanded leaves, which also had the lowest content of Mg among all the leaves of the rosette. The oldest leaves maintained a higher Mg content. [¹⁴C]Sucrose labelling showed that sucrose export from the uppermost expanded leaves was inhibited. In contrast, sucrose export from the oldest leaves, which are close to, and export mainly to, the roots, was not restricted. In response to Mg deficiency, the BvSUT1 gene encoding a companion cell sucrose/H⁺ symporter was induced in the uppermost expanded leaves, but without further enhancement of sucrose loading into the phloem. The observed increase in BvSUT1 gene expression supports the idea that sucrose loading into the phloem is defective, resulting in its accumulation in the leaf.     

Quantitative microbial indices in biogas and raw cattle slurries

Biogas slurry, the secondary product of the anaerobic digestion process, is increasingly being used as fertilizer. Information is available on its chemical and physical properties and their effects on plant growth. However, there is a demand to characterize the microbial quality of slurries, which may control further mineralization processes after application to soil. In this study, biogas and raw slurries obtained from six farms were analyzed for their ergosterol and amino sugar concentrations as indices for microbial biomass. A reliable, precise method for determining ergosterol in slurries is presented. Biogas slurries contained significantly less ergosterol (?34%), muramic acid (MurN; ?42%), galactosamine (GalN; ?32%), and fungal glucosamine (GlcN; ?40%) than raw slurries. The mean fungal GlcN to ergosterol ratio (50) and also the mean fungal carbon (C) to bacterial C ratio (0.29) did not significantly differ between the slurry types. The mean microbial C concentration in the biogas slurries was significantly lower than in the raw slurries. Consequently, the contribution of microbial C to slurry organic C was 3.6% in the biogas slurries and 5.7% in the raw slurries. Microbial C revealed significant nonlinear relationships with the fiber and ash concentration, pH, as well as the C/N ratio of the slurries.     

Fine measurement of ergosterol requirements for growth of Saccharomyces cerevisiae during alcoholic fermentation

Yeasts can incorporate a wide variety of exogenous sterols under strict anaerobiosis. Yeasts normally require oxygen for growth when exogenous sterols are limiting, as this favours the synthesis of lipids (sterols and unsaturated fatty acids). Although much is known about the oxygen requirements of yeasts during anaerobic growth, little is known about their exact sterol requirements in such conditions. We developed a method to determine the amount of ergosterol required for the growth of several yeast strains. We found that pre-cultured yeast strains all contained similar amounts of stored sterols, but exhibited different ergosterol assimilation efficiencies in enological conditions [as measured by the ergosterol concentration required to sustain half the number of generations attributed to ergosterol assimilation (P₅₀)]. P₅₀ was correlated with the intensity of sterol synthesis. Active dry yeasts (ADYs) contained less stored sterols than their pre-cultured counterparts and displayed very different ergosterol assimilation efficiencies. We showed that five different batches of the same industrial Saccharomyces cerevisiae ADY exhibited significantly different ergosterol requirements for growth. These differences were mainly attributed to differences in initial sterol reserves. The method described here can therefore be used to quantify indirectly the sterol synthesis abilities of yeast strains and to estimate the size of sterol reserves.     

Study of the biosynthesis of fumonisins B1, B2 and B3 by different strains of Fusarium moniliforme

The relationship between fungal growth and the production of fumonisin on maize grain by 25 strains of Fusarium moniliforme of different origins has been investigated. Although sporulation was essentially the same for all the strains (about 10⁸ propagules g⁻¹ dry matter), ergosterol assays revealed marked variations in fungal biomass. All strains studied produced highly variable amounts of fumonisin B1, the highest levels being observed in strains of ergosterol content above 400 μg g⁻¹. However, no correlation could be established between the synthesized biomass and the quantity of fumonisins produced. We verified that ergosterol is an indicator of mycelial growth, and therefore of the potential toxicity of the analysed grain.     

On-line fluorescence measurements in assessing culture metabolic activities

Summary NADH fluorescence aided by a stoichiometric metabolic pathway model and culture dynamics was used to elucidate the unobservable intracellular physiological state in two metabolically different phases during culture of Clostridium acetobutylicum. The validity of the theoretical model was examined over a range of culture pH regimes and initial sugar concentrations. The H₂/CO₂ gas concentration ratio was found to be an important process parameter. NADH fluorescence detection was compared with simultaneous enzymatic measurements. The specific fluorescence (fluorescence per biomass, F/X) provided a distinction between oxidative and reductive culture metabolism independent of the pH or substrate concentration changes. A good indicator of the type of culture activity proved to be the dF/dt parameter. The net fluorescence measurements correlated with butanol accumulation under all growth conditions suggesting the possible use of the fluorescence probe as a butanol probe in this fermentation.     

The Pectinase produced by Tubercularia vulgaris in submerged culture using pectin or orange-pulp pellets as inducer

The growth of four strains of the shiitake mushroom Lentinus edodes in solid substrate fermentation in synthetic oak sawdust logs was studied over a 14-week period. Total extracellular phenol oxidase activity and soluble protein were monitored and biomass estimated as the ergosterol content of the fermented sawdust. It was observed that two of the strains had a similar pattern of phenol oxidase activity with two cycles with maxima at 2 and 8 weeks of mycelial growth prior to fruiting. With the other two strains there was a maximum at week 4. For each strain, phenol oxidase activity increased with the cold shock used to induce fruiting. Phenol oxidase activity was not found to be correlated with either soluble protein or total fungal biomass in the fermented sawdust, which were correlated for each strain. Quantification of biomass from submerged liquid culture on the basis of dry weight and ergosterol contents showed that the strains fell into the same two groups with respect to the ergosterol to biomass ratio, which was markedly lower than that for a strain of L. lepideus.Correspondence to: B. C. Okeke     

Polygalacturonase,biomass and ascospore production by Byssochlamys fulva. II. Effects of sugars found in fruits

Polygalacturonase, biomass, and ascospore production by four strains of Byssochlamys fulva cultured in laboratory media supplemented with glucose, sucrose, or fructose was studied over a 20-day incubation period at 30° C. The production of polygalacturonase was variable, but most activity was detected between 4 and 8 days in 1% sugar media at an initial pH of 4 or 5. The rate of biomass production was retarded early in the incubation period in media initially at pH 3 or 4 as compared to pH 5, but the amount of growth was about the same in media containing the test sugars after 20 days. Large numbers of ascospores were produced between 8 and 10 days in media containing 5% sugar initially at pH 5 and 4. Production of ascospores was retarded at pH 3 in media containing 5% sugar as compared to media initially at pH 5 and 4.     

Chitin and ergosterol combined to measure total and living fungal biomass in ectomycorrhizas

We have studied the chitin and ergosterol contents of ectomycorrhizal roots in three sets of experiments to evaluate them as indicators of fungal biomass. The first set of experiments showed that ageing had a marked effect on ergosterol concentrations. The ergosterol content of 7-month-old, brown, shrunken Pinus sylvestris L.– Paxillus involutus (Fr.) Fr. mycorrhizas was found to be only 10% of that found in white, turgid, 1- or 4-month-old specimens. This supports the hypothesis that the compound is a good indicator of living fungal biomass. Ageing had a lesser effect on chitin concentrations since the chitin levels found in 7-month-old mycorrhizas were still 60% of the levels found in 1- and 4-month-old specimens.
Consequently, the chitin:ergosterol ratio increased from about 14 to 19 in 1- and 4-month-old mycorrhizas respectively to about 110 in 7-month-old mycorrhizas. In the second set of experiments, we found that variation in plant growth had no effect on the chitin:ergosterol ratio in whole root systems of either Alnus incana (L.) Moench or Pinus sylvestris mycorrhizal with Paxillus involutus . In the third set of experiments, we found a constant relationship between the two marker concentrations in 10-month-old root systems of Pinus sylvestris , regardless of fungal species involved, using Paxillus involutus , Piloderma croceum Erikss. & Hjorts and Suillus variegatus (Fr.) O. Kuntze as test organisms. Taken together, the results of this study suggest that both chitin and ergosterol give reliable, but different, relative measures of fungal biomass in mycorrhizal roots. Furthermore, we demonstrate that, in combination, the two chemical markers can be used to estimate both total and living fungal biomass (derived from the chitin:ergosterol ratio).     

RNA/DNA ratio as a growth indicator of stream periphyton

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Quantifying mold biomass on gypsum board: comparison of ergosterol and beta-N-acetylhexosaminidase as mold biomass parameters

Two mold species, Stachybotrys chartarum and Aspergillus versicolor, were inoculated onto agar overlaid with cellophane, allowing determination of a direct measurement of biomass density by weighing. Biomass density, ergosterol content, and beta-N-acetylhexosaminidase (3.2.1.52) activity were monitored from inoculation to stationary phase. Regression analysis showed a good linear correlation to biomass density for both ergosterol content and beta-N-acetylhexosaminidase activity. The same two mold species were inoculated onto wallpapered gypsum board, from which a direct biomass measurement was not possible. Growth was measured as an increase in ergosterol content and beta-N-acetylhexosaminidase activity. A good linear correlation was seen between ergosterol content and beta-N-acetylhexosaminidase activity. From the experiments performed on agar medium, conversion factors (CFs) for estimating biomass density from ergosterol content and beta-N-acetylhexosaminidase activity were determined. The CFs were used to estimate the biomass density of the molds grown on gypsum board. The biomass densities estimated from ergosterol content and beta-N-acetylhexosaminidase activity data gave similar results, showing significantly slower growth and lower stationary-phase biomass density on gypsum board than on agar.     

Switching the mode of sucrose utilization by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H⁺ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.     

Ergosterol and Fusarium mycotoxins content in two maize cultivars under different forms of nitrogen fertilizers

Mycotoxins are secondary metabolites produced by many filamentous fungi, such as Fusarium, and are widespread in nature and can adversely affect almost all organisms. Mycotoxins can be formed in very different conditions such as when plants are growing or in crops that are stored in improper conditions. One of the methods to prevent the formation of mycotoxins in powdered feed is using varieties of appropriate quality and proper mineral fertilization, primarily nitrogen. In this study, the concentration of ergosterol and mycotoxins in the kernels of two maize cultivars from different maturity groups in Poland during the harvest of 2011 and 2012 was investigated using HPLC methods. We aimed to analyse the concentration of ergosterol as a fungal biomass indicator and mycotoxins from two maize cultivars under six different forms of nitrogen fertilizers. The “stay‐green” hybrid ES Paroli was characterized by a significantly lower ergosterol and the ability to accumulate the mycotoxins, compared to the classic cultivar ES Palazzo. The varieties of maize that remain green for a longer time period, the so‐called stay‐green type, are characterized by a different coefficient of nitrogen remobilization compared to the classic hybrids. Their dynamics of growth and accumulation of nutrients indicates a nitrogen fertilization system, indicating that slow‐release fertilizers are potentially more adapted to the cycles of maize vegetation. Hence, compared to the classic variant, the “stay‐green” variety uses nitrogen more effectively from mineral fertilizers. Such hybrids are healthier and remain in good condition for a longer time; therefore, they are characterized by a lower pathogen pressure. Thus, the cultivation of such varieties can be considered as one of the elements of integrated maize production. We observed identical reactions of the studied experimental factors for both vegetation seasons, which indicates the lack of environmental impact on the functioning and interaction of experimental factors.