The effects of environmental factors on the synthesis of water-soluble Monascus red pigments via submerged fermentation: a review.

Monascus pigments (MPs) have been used as natural food pigments for many years. There is a high demand for Monascus red pigments (MRPs) to enhance color and for antibacterial and cancer prevention therapies in food and medicine. Most MRPs are not water soluble, and the yield of water-soluble MRPs is naturally low. On the other hand, water-soluble MRP is more cost effective for application in industrial mass production. Therefore, it is important to improve the yield of water-soluble MRPs. Environmental factors have a significant influence on the synthesis of water-soluble MRPs, which is crucial for the development of industrial production of water-soluble MRPs. This review introduces the biosynthetic pathways of water-soluble MRPs and summarizes the effects of environmental factors on the yield of water-soluble MRPs. Acetyl coenzyme A (acetyl-CoA) is a precursor for MPs synthesis. Carbon and nitrogen sources and the carbon/nitrogen ratio can impact MP production by regulating the metabolic pathway of acetyl-CoA. Optimization of fermentation conditions to change the morphology of Monascus can stimulate the synthesis of MPs. The appropriate choice of nitrogen sources and pH values can promote the synthesis of MRPs from MPs. Additives such as metal ions and non-ionic surfactants can affect the fluidity of Monascus cell membrane and promote the transformation of MRPs into water-soluble MRPs. This review will lay the foundation for the industrial production of water-soluble MRPs. © 2024 Society of Chemical Industry.

Nucleic acid demethylase MpAlkB1 regulates the growth, development, and secondary metabolite biosynthesis in Monascus purpureus.

Nucleic acid demethylases of α-ketoglutarate-dependent dioxygenase (AlkB) family can reversibly erase methyl adducts from nucleobases, thus dynamically regulating the methylation status of DNA/RNA and playing critical roles in multiple cellular processes. But little is known about AlkB demethylases in filamentous fungi so far. The present study reports that Monascus purpureus genomes contain a total of five MpAlkB genes. The MpAlkB1 gene was disrupted and complemented through homologous recombination strategy to analyze its biological functions in M. purpureus. MpAlkB1 knockout significantly accelerated the growth of strain, increased biomass, promoted sporulation and cleistothecia development, reduced the content of Monascus pigments (Mps), and strongly inhibited citrinin biosynthesis. The downregulated expression of the global regulator gene LaeA, and genes of Mps biosynthesis gene cluster (BGC) or citrinin BGC in MpAlkB1 disruption strain supported the pleiotropic trait changes caused by MpAlkB1 deletion. These results indicate that MpAlkB1-mediated demethylation of nucleic acid plays important roles in regulating the growth and development, and secondary metabolism in Monascus spp.

Volatile Organic Compound-Mediated Antifungal Activity of Pichia spp. and Its Effect on the Metabolic Profiles of Fermentation Communities.

Volatile organic compounds (VOCs) are chemicals responsible for antagonistic activity between microorganisms. The impact of VOCs on microbial community succession of fermentation is not well understood. In this study, Pichia spp. were evaluated for VOC production as a part of antifungal activity during baijiu fermentation. The results showed that the abundance of Pichia in the defect group (agglomerated fermented grains) was lower than that in control group, and a negative interaction between Pichia and Monascus was determined (P < 0.05). In addition, the disruption of fungi was significantly related to the differences of metabolic profiles in fermented grains. To determine production of VOCs from Pichia and its effect on Monascus purpureus, a double-dish system was assessed, and the incidence of M. purpureus reduction was 39.22% after 7 days. As to antifungal volatile compounds, 2-phenylethanol was identified to have an antifungal effect on M. purpureus through contact and noncontact. To further confirm the antifungal activity of 2-phenylethanol, scanning electron microscopy showed that 2-phenylethanol widely and significantly inhibited conidium germination and mycelial growth of filamentous fungi. Metatranscriptomic analysis revealed that the Ehrlich pathway is the metabolic path of 2-phenylethanol in Pichia and identified potential antifungal mechanisms, including protein synthesis and DNA damage. This study demonstrated the role of volatile compound-mediated microbial interaction in microbiome assembly and discovered a plausible scenario in which Pichia antagonized fungal blooms. The results may improve the niche establishment and growth of the functional yeast that enhances the flavor of baijiu.IMPORTANCE Fermentation of food occurs within communities of interacting species. The importance of microbial interactions in shaping microbial structure and metabolic performance to optimize the traditional fermentation process has long been emphasized, but the interaction mechanisms remain unclear. This study applied metabolome analysis and amplicon sequencing along with metatranscriptomic analysis to examine the volatile organic compound-mediated antifungal activity of Pichia and its effect on the metabolism of ethanol during baijiu fermentation, potentially enhancing the establishment of the fermentation niche and improving ethanol metabolism.

Evaluating the effects of microparticle addition on mycelial morphology, natural yellow pigments productivity, and key genes regulation in submerged fermentation of Monascus purpureus.

Morphology plays an important role in fungal fermentation and secondary metabolites biosynthesis. One novel technique, microparticle-enhanced cultivation was successfully utilized to control the morphology of Monascus purpureus precisely and enhance the yield of yellow pigments. The production of yellow pigments increased to 554.2 U/ml when 4 g/L 5000 mesh talc added at 24 h. Field emission scanning electron microscope observation indicated that the actual effect depends on the properties of microparticle. Sharp-edged microparticles showed better stimulatory effects than smooth, round-shaped ones. Particle size analysis, scanning electron microscope, and cell integrity evaluation proved obvious morphological changes were induced by talc addition, including smaller mycelial size, rougher hyphae, and decreased cell integrity. Furthermore, the expression levels of MrpigG, MrpigD, MrpigE, and MrpigH were significantly upregulated by the addition of talc. It indicated that the microparticle could not only affect the mycelial morphology, but also influence the expression levels of key genes in biosynthetic pathway of Monascus yellow pigments.

Promotion of monacolin K production in Monascus extractive fermentation: the variation in fungal morphology and in the expression levels of biosynthetic gene clusters.

BACKGROUND: Monacolin K, an important secondary metabolite of Monascus, possesses a cholesterol-lowering effect and is widely used in the manufacture of antihypertensive drugs. In the present study, we constructed an extractive fermentation system by adding non-ionic surfactant and acquired a high monacolin K yield. The mechanism was determined by examining both cell morphology and the transcription levels of the related mokA-I genes in the monacolin K biosynthetic gene cluster. RESULTS: The monacolin K yield was effectively increased to 539.59 mg L-1 during extraction, which was an increase of 386.16% compared to that in the control group fermentation. The non-ionic surfactant showed good biocompatibility with Monascus. Electron scanning microscopy revealed alterations in the morphology of Monascus. The loosened mycelial structure and increased number of cell surface wrinkles were found to be related to the increased cell-membrane permeability and extracellular accumulation of monacolin K. Gene expression levels were measured via a quantitative reverse transciptase-polymerase chain reaction. By contrast, in the control group, mokA, mokB, mokC, mokD and mokF showed higher-level and longer-term expression in the extractive fermentation group, whereas mokE and mokG did not present a similar trend. The expression levels of mokH and mokI, encoding a transcription factor and efflux pump, respectively, were also higher than the control levels. CONCLUSION: The addition of a non-ionic surfactant to Monascus fermentation effectively increases the yield of monacolin K by transforming the fungus morphology and promoting the expression of monacolin K biosynthesis genes. © 2021 Society of Chemical Industry.

Overexpression of global regulator LaeA increases secondary metabolite production in Monascus purpureus.

Monascus is a filamentous fungus that produces several secondary metabolites. Here, we investigated the effects of the global regulator LaeA on the synthesis of pigments and monacolin K in Monascus purpureus with spectrophotometer and HPLC methods. The LaeA gene was isolated from M. purpureus M1 to create an overexpression construct. An LaeA-overexpressing strain L3 was with 48.6% higher monacolin K production than the M1 strain. The L3 strain also produced higher Monascus pigments than the M1 strain. SEM showed that LaeA overexpression resulted in altered mycelial morphology. Compared with the M1 strain, the L3 strain expressed higher levels of monacolin K synthesis-related genes mokA, mokB, mokE, and mokH. Overall, these results suggest that LaeA plays a role in regulating the production of secondary metabolites and mycelial growth in Monascus. This study provides important insights into the mechanisms underlying the effects of the LaeA gene on the secondary metabolites of M. purpureus.

Evaluation of tyrosol and farnesol as inducer in pigment production by Monascus purpureus ATCC16365.

This study focused on investigating the effect of exogenously applied two quorum sensing molecules (tyrosol and farnesol) on the synthesis of bioactive metabolites (pigments, lactic acid, ethanol, and citric acid) in Monascus purpureus ATCC16365. None of the tested concentrations (62.5, 125, 250, and 500 µl/L) of farnesol affected the synthesis of metabolites as well as cell growth. As with farnesol application, none of the tested concentrations (3.45, 6.9, 13.8, and 27.6 mg/L) of tyrosol caused a significant change in the synthesis of lactic acid and citric acid as well as cell growth. Conversely, all of the tested concentrations of tyrosol increased pigment synthesis but reduced ethanol synthesis, compared with the control. Maximum increases (3.16-, 2.68-, and 2.87-fold increase, respectively) in yellow, orange, and red pigment production were achieved, especially when 6.9-mg/L tyrosol was added to the culture on day 3. On the contrary, 6.9-mg/L tyrosol reduced the content of citrinin by approximately 51.5%. This is the first report on the effect of tyrosol and farnesol on the synthesis of Monascus metabolites. Due to potential properties, such as low price, nonhuman toxicity, promotion of pigment synthesis, and reduction in citrinin synthesis, tyrosol can be used as a novel inducer in the fermentative production of Monascus pigments.

Effect of nonionic surfactant Brij 35 on morphology, cloud point, and pigment stability in Monascus extractive fermentation.

BACKGROUND: Nonionic surfactant Brij 35 in submerged fermentation of Monascus can significantly increase Monascus pigment yield. Here, the effects of nonionic surfactant Brij 35 on Monascus pigment secretion in extractive fermentation are discussed in terms of cell morphology, cloud point change, and pigment stability. RESULTS: At Brij 35 concentrations up to 32 g L-1 , the higher concentrations led to the loosening of the network structure on the surface of the fungal wall, enhanced cell wall permeability, and increased abundance of lipid droplets. Alternatively, when the concentration of Brij 35 exceeded 32 g L-1 , a large amount of substances accumulated on the surface of the fungal wall, permeability reduced, and the degree of oil droplet dispersion in cells decreased. Further, during extractive fermentation, Brij 35 induced formation of a grid structure on the fungal wall surface beginning on day 2, increased the number of intracellular lipid droplets, and promoted intracellular pigment secretion into the extracellular environment. When the cloud point temperature in the fermentation system approached that of fermentation, the nonionic surfactant exhibited stronger Monascus pigment extraction capacity, thereby enhancing pigment yield. Hence, Brij 35 can improve pigment stability and effectively reduce damage caused by natural factors, such as light and temperature. CONCLUSION: Brij 35 promotes the secretion of pigment by changing the fungal wall structure and cloud point, as well as by improving pigment stability. © 2020 Society of Chemical Industry.

Acidic conditions induce the accumulation of orange Monascus pigments during liquid-state fermentation of Monascus ruber M7.

The influence of pH on the biosynthesis of orange Monascus pigments (OMPs) in Monascus ruber M7 was investigated. Under acidic fermentation conditions, pigment mixtures predominantly rich in OMPs were obtained. HPLC analysis revealed the presence of four orange components (O1-O4) and four yellow components (Y1-Y4) in the mixtures, and the dominant ones were O1 and O3, which accounted for 56.0% to 75.9% of the total pigments in the pH range 3-6. Subsequently, O1 and O3 were identified by LC-DAD-ESI/MS as Rubropunctatin and Monascorubrin, respectively. The yield of OMPs was observed to be inversely dependent on pH. At pH 3, large amounts of OMPs with high purity (79.1%) were accumulated. A real-time quantitative PCR analysis revealed that the expression of genes related to the biosynthesis of OMPs in M. ruber M7 was upregulated at acidic pH as compared to neutral pH, and the variation in the level of expression of these genes with pH was consistent with the production of OMPs. These results indicated that the large accumulation of OMPs under acidic condition involved the acidic pH-induced transcription of genes related to the biosynthesis of OMPs. These results would contribute towards the development of an efficient technology for large-scale production of OMPs.

Variations in Monascus pigment characteristics and biosynthetic gene expression using resting cell culture systems combined with extractive fermentation.

Monascus pigments are promising sources of natural food colorants, and their productivity can be improved by a novel extractive fermentation technology. In this study, we investigated the variations in pigment characteristics and biosynthetic gene expression levels in resting cell culture systems combined with extractive fermentation in Monascus anka GIM 3.592. Although the biomass was low at about 6 g/L DCW, high pigment titer of approximately 130 AU470 was obtained in the resting culture with cells from extractive fermentation, illustrating that it had a good biocatalytic activity for pigment synthesis. The oxidation-reduction potential value correlated with the rate of relative content of the intracellular orange pigments to the yellow pigments (O/Y, r > 0.90, p < 0.05), indicating that the change in pigment characteristics may be responsible for the cellular redox activity. The up- or down-regulation of the pigment biosynthetic genes (MpFasA2, MpFasB2, MpPKS5, mppD, mppB, mppR1, and mppR2) in the resting culture with extractive culture cells was demonstrated by real-time quantitative polymerase chain reaction analysis. Moreover, the mppE gene associated with the yellow pigment biosynthesis was significantly (p < 0.05) down-regulated by about 18.6%, whereas the mppC gene corresponding to orange pigment biosynthesis was significantly (p < 0.05) up-regulated by approximately 21.0%. These findings indicated that extractive fermentation was beneficial for the biosynthesis of the intracellular orange pigment. The mechanism described in this study proposes a potential method for the highly efficient production of Monascus pigments.

Pigment fingerprint profile during extractive fermentation with Monascus anka GIM 3.592.

BACKGROUND: Traditional submerged fermentation mainly accumulates intracellular orange pigments with absorption maxima at 470 nm, whereas extractive fermentation of Monascus spp. with Triton X-100 can promote the export of intracellular pigments to extracellular broth, mainly obtaining extracellular yellow pigments with absorption maxima at approximately 410 nm. In this study, a strain of Monascus (M. anka GIM 3.592) that produces high yields of pigments was employed to investigate the differences in pigment fingerprint profiles between submerged and extractive fermentations. RESULTS: Using extractive fermentation with this high-yield strain, the extracellular pigments exhibited an absorption maximum at 430 nm, not 410 nm, as previously observed. By comparing the pigment fingerprint profiles between submerged and extractive fermentations, extractive fermentation was found to not only export intracellular pigments to the extracellular broth, but also to form four other yellow pigments (Y1-Y4) that accounted for a large proportion of the extracellular pigments and that were not produced in submerged fermentation. The yields of Y1-Y4 were closely related to the concentration and feeding time point of Triton X-100. Y1-Y4 presented identical UV-Vis spectra with absorption maxima at 430 nm and fluorescence spectra with absorption maxima (emission) at 565 nm. HPLC-MS and the spectral analysis showed that the four pigments (Y1-Y4) had not been previously reported. The results indicated that these pigments may rely on the bioconversion of orange pigments (rubropunctatin and monascorubrin). CONCLUSIONS: Using extractive fermentation with M. anka led to a high yield of extracellular yellow pigments (AU410 nm = 114), and the pigment fingerprint profile significantly differed compared to the results of traditional submerged fermentation. These results provide information and a detailed view of the composition and variation of pigments in extractive fermentation and could also contribute to characterizing pigment metabolism during extractive fermentation.

Kinetic of orange pigment production from Monascus ruber on submerged fermentation.

Pigments produced by species of Monascus have been used to coloring rice, meat, sauces, wines and beers in East Asian countries. Monascus can produce orange (precursor), yellow and red pigments. Orange pigments have low solubility in culture media and when react with amino groups they become red and largely soluble. The orange pigments are an alternative to industrial pigment production because the low solubility facilitates the downstream operations. The aim of this work was to study the kinetic on the production of orange pigments by Monascus ruber CCT 3802. The shaking frequency of 300 rpm was favorable to production, whereas higher shaking frequencies showed negative effect. Pigment production was partially associated with cell growth, the critical dissolved oxygen concentration was between 0.894 and 1.388 mgO2 L-1 at 30 °C, and limiting conditions of dissolved oxygen decreased the production of orange pigments. The maintenance coefficient (mo) and the conversion factor of oxygen in biomass (Yo) were 18.603 mgO2 g x-1  h-1 and 3.133 gx gO 2-1 and the consideration of these parameters in the oxygen balance to estimate the biomass concentration provided good fits to the experimental data.

Effects and Mechanism of Blue Light on Monascus in Liquid Fermentation.

The effect of light on Monascus and the underlying mechanism have received a great deal of interest for the industrial application of Monascus pigments. In this study, we have examined the effects of blue light on the culture morphology, mycelium growth, pigments, and citrinin yield of Monascus in liquid-state and oscillation fermentation, and explored the mechanism at a physiological level. It was found that blue light affected the colony morphology, the composition (chitin content), and permeability of the Monascus mycelium cell wall in static liquid culture, which indicates blue light benefits pigments secreting from aerial mycelium to culture medium. In liquid oscillation fermentation, the yields of Monascus pigments in fermentation broth (darkness 1741 U/g, blue light 2206 U/g) and mycelium (darkness 2442 U/g, blue light 1900 U/g) cultured under blue light and darkness are different. The total pigments produced per gram of Monascus mycelium under blue light was also higher (4663 U/g) than that in darkness (4352 U/g). However, the production of citrinin (88 µg/g) under blue light was evidently lower than that in darkness (150 µg/g). According to the degradation of citrinin caused by blue light and hydrogen peroxide, it can be concluded that blue light could degrade citrinin and inhibit the catalase activity of Monascus mycelium, subsequently suppressing the decomposition of hydrogen peroxide, which is the active species that degrades citrinin.

Comparative analysis of genetic polymorphisms among Monascus strains by ISSR and RAPD markers.

BACKGROUND: The genus Monascus includes several species of fungi valued across Asia for their culinary uses and diverse medicinal properties. In this study, we evaluated the applicability of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers in characterizing the genetic diversity in 41 Monascus strains collected from various regions of Fujian Province, the leading producer of Monascus in China. RESULTS: Seven screened ISSR primers generated 56 polymorphic bands, of which 93.33% were polymorphic. The genetic similarity coefficients (GSC) of the strains ranged from 0.50 to 1.00. Comparative sequence analysis using seven screened RAPD primers amplified a total of 49 polymorphic bands, of which 81.67% were polymorphic; GSC values ranged from 0.62 to 1.00. CONCLUSION: Correlation analysis revealed a significant positive correlation in genetic distances assessed using above two markers, which indicated they were suitable for Monascus species characterization. ISSR markers were more suitable for the classification and determination of Monascus species, while RAPD markers appear to be preferable for analyzing the differences among strains within the same species. Our study revealed that Monascus possesses rich genetic diversity, and that the genetic relationships among the selected strains were, to a very limited extent, correlated to their geographical variation. © 2016 Society of Chemical Industry.

Releasing intracellular product to prepare whole cell biocatalyst for biosynthesis of Monascus pigments in water-edible oil two-phase system.

Selective releasing intracellular product in Triton X-100 micelle aqueous solution to prepare whole cell biocatalyst is a novel strategy for biosynthesis of Monascus pigments, in which cell suspension culture exhibits some advantages comparing with the corresponding growing cell submerged culture. In the present work, the nonionic surfactant Triton X-100 was successfully replaced by edible plant oils for releasing intracellular Monascus pigments. High concentration of Monascus pigments (with absorbance nearly 710 AU at 470 nm in the oil phase, normalized to the aqueous phase volume approximately 142 AU) was achieved by cell suspension culture in peanut oil-water two-phase system. Furthermore, the utilization of edible oil as extractant also fulfills the demand for application of Monascus pigments as natural food colorant.

The pigment characteristics and productivity shifting in high cell density culture of Monascus anka mycelia.

BACKGROUND: Monascus mycelia and pigments are promising sources of food and medicine with their potential pharmaceutical values and health-improving functions. Using high cell density fermentation of Monascus spp. to achieve higher mycelium and yellow pigment production is worthy to be researched. In this study, the characteristics and productivity shifting of pigments in high cell density culture of Monascus anka GIM 3.592 were investigated. RESULTS: The high yield of Monascus mycelia up to 39.77 g/L dry cell weight (DCW), which was achieved by fed-batch fermentation with the feeding medium containing C, N, P and trace elements, was four times higher than that of conventional batch culture. But the total pigment production decreased by 14.6 %, which suggested non-coupled growth. Potential novel yellow pigments accumulated constantly at the late stage of the fed-batch culture, which resulted in a shift in pigment characteristics so that yellow pigments became the dominant pigments. Citrinin production was extremely low and independent of feeding ingredients. CONCLUSIONS: This study provided a suitable fermentation strategy to produce functional Monascus mycelia with a high proportion of yellow pigments in high cell density culture. For the first time, it reported the pigment productivity and characteristics shifting in high cell density culture of Monascus.

Accumulation of yellow Monascus pigments by extractive fermentation in nonionic surfactant micelle aqueous solution.

Monascus species can produce various secondary metabolites of polyketide structure. In the current study, it is found that an interesting phenomenon, i.e., submerged culture of Monascus species in an aqueous solution majorly accumulated intracellular orange Monascus pigments exhibiting one peak at 470 nm with absorbance of 32 OD while extractive fermentation in a nonionic surfactant micelle aqueous solution produced extracellular and intracellular yellow Monascus pigments exhibiting one peak at 410 nm with absorbance 30 OD and 12 OD, respectively. The spectrum profiles of both intracellular and extracellular Monascus pigments were affected by surfactant loading, extractive fermentation time, and surfactant adding time. Meanwhile, the instability of orange Monascus pigments in the extracellular nonionic surfactant micelle aqueous solution was also confirmed experimentally. The mechanism behind this phenomenon is attributed to the export of intracellular yellow Monascus pigments into its broth by extractive fermentation. The transferring of intracellular yellow Monascus pigments into its broth blocks yellow Monascus pigments from further enzymatic conversion or eliminates the feedback inhibition of yellow Monascus pigments based on the biosynthetic pathway of Monascus pigments.

Stimulatory effects of blue light on the growth, monascin and ankaflavin production in Monascus.

Light is an important signal for fungi. We analyzed the influence of blue light of various intensities and illumination times on growth, monascin (MS) and ankaflavin (AK) biosyntheses in Monascus strain M9. Blue light changed the color of colonies. The colonies grown in the dark were orange, but turned pale when exposed to continuous blue light. MS production increased by 12.5, 27, and 14.5 % under blue light of 100 lux for 15 min/day, 100 lux for 30 min/day, and 200 lux for 15 min/day, respectively, compared to growth in the dark. AK production increased by 14.4, 22, and 13 % under the same condition. MS and AK production decreased when exposed to blue light of 300 and 450 lux. The expression of pigment biosynthetic genes were analyzed by real-time quantitative PCR and correlated with phenotypic production of MS and AK.

Enhanced production of Monacolin K by addition of precursors and surfactants in submerged fermentation of Monascus purpureus 9901.

The main problem in Monacolin K (MK) production by submerged fermentation of Monascus purpureus is low productivity. In this study, on one hand, addition of precursors was used to activate the biosynthesis of MK. When 4.0 g/L of sodium citrate was supplemented at the 48th H of the fermentation, the final MK production reached to 1,658.9 ± 28.5 mg/L after 20 day of fermentation, which was improved by 52.6% compared with that of the control. On the other hand, addition of surfactants could increase the permeability of cell membrane, thus driving more intracellular metabolites secreted into the fermentation broth and alleviating the product inhibition. When 40.0 g/L of Triton X-100 was added at the beginning of the fermentation, the final MK production reached to 2,026.0 ± 30.4 mg/L after 20 day of fermentation, which was improved by 84.9% compared with that of the control. These results are helpful to provide some new insights into the biosynthetic regulation on MK production; the approach can be applied to other fungal fermentation processes for enhancing production of useful metabolites.

Quantification of the glucosamine content in the filamentous fungus Monascus ruber cultured on solid surfaces.

We evaluated whether the glucosamine content in the filamentous fungus Monascus ruber NBRC 32318, cultured on a solid surface (agar) containing different carbon and nitrogen sources, could be used as a measure of biomass. The relationship between the amounts of glucosamine and biomass was independent of the cultivation period, but was dependent on the carbon source (D-glucose, D-fructose, maltose, sucrose, or rice starch) and the nitrogen source (ammonium chloride, sodium nitrate, monosodium glutamate, or yeast extract) in the agar; it was also dependent on the culture method (solid-surface culture or submerged culture). We concluded that the amount of glucosamine extracted from M. ruber is a useful index for the fungal biomass when the relationship between M. ruber biomass and glucosamine content has previously been calibrated for the carbon and nitrogen sources used. Examination of microphotographs of M. ruber hyphae in conjunction with quantification of the glucosamine and biomass contents indicated that the variation in the glucosamine content per unit biomass affects the hyphal morphology of the fungus, and especially the hyphal diameter.