Acid-precipitable Polymeric Lignin Research Articles

Screening and Identification of some APPL (Acid Precipitable Polymeric Lignin) Producing Streptomycetes

Thirty seven actinomycetes isolates were isolated from Damietta soil using starch nitrate agar media. These isolates were screened for APPL (acid precipitable polymeric lignin) by using minimal salts-yeast extract medium with rice and wheat straws. Three actinomycetes isolates showed high APPL production. These isolates also showed production of cellulase and xylanase with different levels. These isolates were identified by using morphological, physiological and chemotaxonomy criteria and were identified as Streptomyces canus, Streptomyces libani and Streptomyces cyaneus.

Laccase SilA from Streptomyces ipomoeae CECT 3341, a key enzyme for the degradation of lignin from agricultural residues?

The role of laccase SilA produced by Streptomyces ipomoeae CECT 3341 in lignocellulose degradation was investigated. A comparison of the properties and activities of a laccase-negative mutant strain (SilA−) with that of the wild-type was studied in terms of their ability to degrade lignin from grass lignocellulose. The yields of solubilized lignin (acid precipitable polymeric lignin, APPL) obtained from wheat straw by both strains in Solid State Fermentation (SSF) conditions demonstrated the importance of SilA laccase in lignin degradation with the wild-type showing 5-fold more APPL produced compared with the mutant strain (SilA−). Analytical pyrolysis and FT-IR (Fourier Transform Infrared Spectroscopy) confirmed that the APPL obtained from the substrate fermented by wild-type strain was dominated by lignin derived methoxyphenols whereas those from SilA− and control APPLs were composed mainly of polysaccharides. This is the first report highlighting the role of this laccase in lignin degradation.

Enhanced delignification of steam-pretreated poplar by a bacterial laccase

The recalcitrance of woody biomass, particularly its lignin component, hinders its sustainable transformation to fuels and biomaterials. Although the recent discovery of several bacterial ligninases promises the development of novel biocatalysts, these enzymes have largely been characterized using model substrates: direct evidence for their action on biomass is lacking. Herein, we report the delignification of woody biomass by a small laccase (sLac) from Amycolatopsis sp. 75iv3. Incubation of steam-pretreated poplar (SPP) with sLac enhanced the release of acid-precipitable polymeric lignin (APPL) by ~6-fold, and reduced the amount of acid-soluble lignin by ~15%. NMR spectrometry revealed that the APPL was significantly syringyl-enriched relative to the original material (~16:1 vs. ~3:1), and that sLac preferentially oxidized syringyl units and altered interunit linkage distributions. sLac’s substrate preference among monoaryls was also consistent with this observation. In addition, sLac treatment reduced the molar mass of the APPL by over 50%, as determined by gel-permeation chromatography coupled with multi-angle light scattering. Finally, sLac acted synergistically with a commercial cellulase cocktail to increase glucose production from SPP ~8%. Overall, this study establishes the lignolytic activity of sLac on woody biomass and highlights the biocatalytic potential of bacterial enzymes.

Role of a thermostable laccase produced by Streptomyces ipomoeae in the degradation of wheat straw lignin in solid state fermentation

Streptomycetes are actually considered one of the main groups of ligninolytic microoganisms producing a lignin-carbohydate complex named APPL (acid precipitable polymeric lignin) when growing on lignocellulosic materials. Although in these conditions the production of hemicellulolytic and oxidative extracellular enzymes were reported the specific role of laccases in lignin degradation is poorly understood. SilA, a thermostable salt-resistant and pH-versatile laccase produced by Streptomyces ipomoeae CECT 3341 was recently discovered and their particular characteristics make attractive to deep in its knowledge for biotechnological and environmental purposes. Pyrolysis/GC–MS was used to analyse the behaviour of the laccase-producing strain (SilA strain) and a laccase-negative mutant (SilA− strain) when growing on wheat straw in solid-state fermentation (SSF). Quantitative yields of APPL and the relative abundance of lignin-derived compounds were much higher for SilA strain than for SilA− showing a higher solubilizing activity of S. ipomoeae wild-type on lignocellulosic residues. Nonetheless the patterns of lignin derived compounds found in the APPL pyrograms were similar for both strains and distinct from the control showing a shortening of lignin propyl side-chains. Our results demonstrate that SilA laccase is a key enzyme in the lignin solubilization by S. ipomoeae and also points to the involvement of other oxidative enzymatic activities distinct to laccase in this process.

DuPont and Quad County Corn Processors sign multi-year enzyme supply contract for cellulosic biofuel

Rice straw is the most abundant agricultural residue on a global scale and is widely available as feedstock for cellulosic fuel production. However, it is highly recalcitrant to biochemical deconstruction and also generates inhibitors that affect enzymatic saccharification. Rice straw from eastern Arkansas was subjected to dilute acid pretreatment (160 °C, 48 min and 1.0% sulfuric acid) and solid-state fermentation with two lignocellulolytic fungi, Trametes hirsuta and Myrothecium roridum, and their saccharification efficacies were compared. T. hirsuta and M. roridum were tested separately; pretreatment of rice straw with either strain for seven days resulted in 19 and 70% enrichment of its holocellulose content, respectively. However, liquid chromatography analysis of the alkali extracts showed significant differences in cell wall degradation by T. hirsuta and M. roridum. T. hirsuta removed 15% more phenolic compounds and 38% more glucan than M. roridum, while M. roridum removed 77% more xylan than T. hirsuta. Fungal and dilute acid pretreated biomass was then hydrolyzed using Accellerase® 1500, a saccharification cocktail. Saccharification efficiency of M. roridum was 37% higher than that of dilute acid pretreatment of rice straw, requiring 8% lower enzyme loading and 50% shorter enzymatic hydrolysis duration. Alkali extraction of fungal pretreated biomass also yielded 10–15 g kg−1 of acid precipitable polymeric lignin (APPL), which is a valuable co-product for biorefineries. In comparison to dilute acid pretreatment, fungal pretreatment could be a cost-effective alternative for the degradation of recalcitrant biomass, such as rice straw.

Insights into biological delignification of rice straw by Trametes hirsuta and Myrothecium roridum and comparison of saccharification yields with dilute acid pretreatment

Rice straw is the most abundant agricultural residue on a global scale and is widely available as feedstock for cellulosic fuel production. However, it is highly recalcitrant to biochemical deconstruction and also generates inhibitors that affect enzymatic saccharification. Rice straw from eastern Arkansas was subjected to dilute acid pretreatment (160 °C, 48 min and 1.0% sulfuric acid) and solid-state fermentation with two lignocellulolytic fungi, Trametes hirsuta and Myrothecium roridum, and their saccharification efficacies were compared. T. hirsuta and M. roridum were tested separately; pretreatment of rice straw with either strain for seven days resulted in 19 and 70% enrichment of its holocellulose content, respectively. However, liquid chromatography analysis of the alkali extracts showed significant differences in cell wall degradation by T. hirsuta and M. roridum. T. hirsuta removed 15% more phenolic compounds and 38% more glucan than M. roridum, while M. roridum removed 77% more xylan than T. hirsuta. Fungal and dilute acid pretreated biomass was then hydrolyzed using Accellerase® 1500, a saccharification cocktail. Saccharification efficiency of M. roridum was 37% higher than that of dilute acid pretreatment of rice straw, requiring 8% lower enzyme loading and 50% shorter enzymatic hydrolysis duration. Alkali extraction of fungal pretreated biomass also yielded 10–15 g kg−1 of acid precipitable polymeric lignin (APPL), which is a valuable co-product for biorefineries. In comparison to dilute acid pretreatment, fungal pretreatment could be a cost-effective alternative for the degradation of recalcitrant biomass, such as rice straw.

Poly R decolorization and APPL production by Streptomyces violaceoruber and Streptomyces spiroverticillatus.

Two mesophilic streptomycetes (S. violaceoruber and S. spiroverticillatus) were selected to study their Poly R-478 decolorization ability and lignocellulose solubilizing activity. Both strains were able to degrade Poly R-478 dye and ferulic acid during growth on a minimal salts medium. The Poly R-478 decolorizing activities of both strains were induced by adding different carbon sources to the culture media. S. violaceoruber could decolorize 63% of Poly R-478 after 24 h. Both strains could solubilize straw and produce acid-precipitable polymeric lignin (APPL) with different efficiency. From the major extracellular enzymes recovery of both strains on rice and wheat straw, we can predicate that the biodegradation process was partial indicating a possible utilization in biological delignification.

Roles of small laccases from Streptomyces in lignin degradation.

Laccases (EC 1.10.3.2) are multicopper oxidases that can oxidize a range of substrates, including phenols, aromatic amines, and nonphenolic substrates. To investigate the involvement of the small Streptomyces laccases in lignin degradation, we generated acid-precipitable polymeric lignin obtained in the presence of wild-type Streptomyces coelicolor A3(2) (SCWT) and its laccase-less mutant (SCΔLAC) in the presence of Miscanthus x giganteus lignocellulose. The results showed that strain SCΔLAC was inefficient in degrading lignin compared to strain SCWT, thereby supporting the importance of laccase for lignin degradation by S. coelicolor A3(2). We also studied the lignin degradation activity of laccases from S. coelicolor A3(2), Streptomyces lividans TK24, Streptomyces viridosporus T7A, and Amycolatopsis sp. 75iv2 using both lignin model compounds and ethanosolv lignin. All four laccases degraded a phenolic model compound (LM-OH) but were able to oxidize a nonphenolic model compound only in the presence of redox mediators. Their activities are highest at pH 8.0 with a low krel/Kapp for LM-OH, suggesting that the enzymes’ natural substrates must be different in shape or chemical nature. Crystal structures of the laccases from S. viridosporus T7A (SVLAC) and Amycolatopsis sp. 75iv2 were determined both with and without bound substrate. This is the first report of a crystal structure for any laccase bound to a nonphenolic β-O-4 lignin model compound. An additional zinc metal binding site in SVLAC was also identified. The ability to oxidize and/or rearrange ethanosolv lignin provides further evidence of the utility of laccase activity for lignin degradation and/or modification.

Streptomyces griseorubens mediated delignification of paddy straw for improved enzymatic saccharification yields

Biological pretreatment of paddy straw was carried out using an actinomycete isolate, identified as Streptomyces griseorubens ssr38, for delignification under solid state fermentation and enhanced sugar recovery by enzymatic saccharification. After 10days incubation, the inoculated paddy straw was extracted with mild alkali and high absorbance at 205nm was shown by the extracts indicating the ability of S. griseorubens ssr38 to depolymerize/solubilize lignin to a high extent. Also, almost 25% of depolymerized lignin could be recovered as value-added acid-precipitable polymeric lignin (APPL) as compared to controls. Enrichment in carbohydrate content of inoculated paddy straw following delignification led to a high saccharification efficiency of 97.8% upon enzymatic hydrolysis with Accelerase®1500. The study, therefore, proves the potential of actinomycetes, besides the conventionally used white-rot fungi, for biological pretreatment, in the biomass to bioethanol process, with respect to the high extent of delignification, lignin recovery, cellulose enrichment and very high saccharification efficiency.

Biodegradation of high molecular weight lignin under sulfate reducing conditions: Lignin degradability and degradation by-products

This study is designed to investigate the biodegradation of high molecular weight (HMW) lignin under sulfate reducing conditions. With a continuously mesophilic operated reactor in the presence of co-substrates of cellulose, the changes in HMW lignin concentration and chemical structure were analyzed. The acid precipitable polymeric lignin (APPL) and lignin monomers, which are known as degradation by-products, were isolated and detected. The results showed that HMW lignin decreased and showed a maximum degradation capacity of 3.49 mg/l/day. APPL was confirmed as a polymeric degradation by-product and was accumulated in accordance with HMW lignin reduction. We also observed non-linear accumulation of aromatic lignin monomers such as hydrocinnamic acid. Through our experimental results, it was determined that HMW lignin, when provided with a co-substrate of cellulose, is biodegraded through production of APPL and aromatic monomers under anaerobic sulfate reducing conditions with a co-substrate of cellulose.

Thatch biodegradation and antifungal activities of two lignocellulolytic Streptomyces strains in laboratory cultures and in golf green turfgrass.

The use of lignocellulolytic Streptomyces spp. as biological agents, to enhance thatch degradation in turf and to slow its rate of accumulation while controlling fungal growth in the thatch layer, was studied. In flask scale studies, two lignocellulolytic Streptomyces violaceusniger (= hygroscopicus) strains (YCED9 and WYE53) decomposed thatch (> 30% dry weight) over a 12-week incubation period. Biodegradation was accompanied by production of extracellular cellulases, xylanases, and peroxidases. The accumulation of the polymeric, water-soluble lignin degradation intermediate acid, precipitable polymeric lignin (APPL), was also observed. Residual thatch from 12-week-old cultures had an increased lignin-to-carbohydrate ratio, an indication that although lignin was metabolized, carbohydrates were preferential carbon sources for these actinomycetes. A spore-containing soluble dry powder formulation was used as an inoculum in an in situ field experiment. This formulation was maintained in storage at 4 degrees C for over two years without viability loss. Results from the golf green experiment showed that although treated thatch layers in established greens were not appreciably reduced over the course of one summer, the Streptomyces were active and maintained their populations within the thatch, while fungal growth was suppressed as compared to controls. The results show that treatment of turfgrass with these Streptomyces may be useful for the long-term control of fungal populations within the thatch. Longer field studies are required to assess the long-term potential for also controlling thatch build-up and fungal pathogens.

Degradation of lignosulfonated compounds by Streptomyces viridosporus: effect of the culture medium and the nature of the lignosulfonate molecule

Lignosulfonated compounds are by-products of the sulfite pulping of wood and have a higher level of biological resistance than lignin. Degradation of lignosulfonated compounds by Streptomyces viridosporus strain T7A growing on nutritive or chemically defined culture media was quantified. Two lignosulfonated compounds, differing in their molecular mass distribution and chemical structure, were used: lignosulfonate (1–80 kDa) and vanisperse, a sulfonated oxylignin (1–20 kDa). The use of nutritive culture medium (containing Biosoyase) allowed both the growth of S. viridosporus and the production of lignin peroxidase (LiP) leading to in vitro lignocellulose solubilization, but no lignosulfonate degradation was observed. Twenty per cent of the lignosulfonate underwent a molecular transformation, producing non-biodegradable acid precipitable polymeric lignin (lignosulfonate-APPL). When a defined culture medium (containing glycerol as additional organic carbon source) was used, LiP production was growth-associated. Under these conditions with periodic addition of glycerol, 21% of the lignosulfonate was degraded after 21 d of incubation. An inhibitory effect of lignosulfonate on biomass growth was suspected. When using vanisperse with the defined culture medium, a degradation yield of 35% was obtained after 20 d of incubation. S. viridosporus was unable to degrade a large amount of either lignosulfonated compound though significant modifications of the molecular mass distributions were observed by gel permeation chromatography.

Enhanced degradation of lignosulfonated compounds by Streptomyces viridosporus

Degradation of lignosulfonated compounds by S. viridosporus strain T7A on nutritive or mineral culture media was quantified. Two lignosulfonated compounds, differing in their molecular mass distributions and chemical structures, were used: lignosulfonate (1–80 kDa) and vanisperse, a sulfonated oxylignin (1–20 kDa). The use of nutritive culture medium (containing Biosoyase) enhanced both the growth of S. viridosporus and production of lignin peroxidase (LiP) leading to enhanced lignocellulose degradation, but no lignosulfonated compound degradation was observed. A fraction of these compounds underwent a molecular transformation, producing non-biodegradable acid precipitable polymeric lignin (APPL). When a mineral culture medium (containing glycerol as additional organic carbon source) was used, approximately 21 and 35% of lignosulfonate and vanisperse were respectively degraded, with little APPL production. LiP activity was growth-associated and significant modifications of molecular mass distributions of both lignosulfonated compounds were observed. A mixed natural microbial population, collected in an aerated lagoon treating Fluff pulp effluent, was used to evaluate the biodegradability of lignosulfonate products from S. viridosporus cultures. This population was (i) unable to degrade lignosulfonate products, and (ii) decreased the lignosulfonate degradation capacity of S. viridosporus. Antagonistic effects of the mixed population on S. viridosporus were observed. In the light of this, bioaugmentation strategies involving addition of S. viridosporus are unlikely to be successful and alternative degradation strategies need to be developed.

Enhanced degradation of lignosulfonated compounds by Streptomyces viridosporus

Degradation of lignosulfonated compounds by S. viridosporus strain T7A on nutritive or mineral culture media was quantified. Two lignosulfonated compounds, differing in their molecular mass distributions and chemical structures, were used: lignosulfonate (1–80 kDa) and vanisperse, a sulfonated oxylignin (1–20 kDa). The use of nutritive culture medium (containing Biosoyase) enhanced both the growth of S. viridosporus and production of lignin peroxidase (LiP) leading to enhanced lignocellulose degradation, but no lignosulfonated compound degradation was observed. A fraction of these compounds underwent a molecular transformation, producing non-biodegradable acid precipitable polymeric lignin (APPL). When a mineral culture medium (containing glycerol as additional organic carbon source) was used, approximately 21 and 33% of lignosulfonate and vanisperse were respectively degraded, with little APPL production. LiP activity was growth-associated and significant modifications of molecular mass distributions of both lignosulfonated compounds were observed. A mixed natural microbial population, collected in an aerated lagoon treating Fluff pulp effluent, was used to evaluate the biodegradability of lignosulfonate products from S. viridosporus cultures. This population was (i) unable to degrade lignosulfonate products, and (ii) decreased the lignosulfonate degradation capacity of S. viridosporus. Antagonistic effects of the mixed population on S. viridosporus were observed. In the light of this, bioaugmentation strategies involving addition of S. viridosporus are unlikely to be successful and alternative degradation strategies need to be developed.

Chemical characterization by pyrolysis/gas chromatography/mass spectrometry of acid-precipitable polymeric lignin (APPL) from wheat straw transformed by selected streptomyces strains

The possible differential biodegradation of wheat straw performed by different Streptomyces strains was investigated on solid-state fermentation conditions. The modified lignin polymer named acid-precipitable polymeric lignin (APPL), harvested with NaOH or water, from wheat straw transformed by selected Streptomyces strains was tentatively characterized by pyrolysis-gas chromatography-mass spectrometry (PY/GC/MS). For comparative analyzes, lignin-carbohydrate-protein complexes obtained from uninoculated wheat straw were used as controls. Significant differences in the p-hydroxyphenyl: guaiacyl: syringyl(H: G: S) and in the syringyl guaiacyl ( S G ) ratios of the APPLs extracted with water from wheat straw transformed by the strains Streptomyces UAH 23, Streptomyces UAH 52 and Streptomyces viridosporus T7 A were detected compared with the control. While S. UAH 23 produced a preferential degradation of the non-condensed Slignin units, a decrease in the condensed G-units was produced by S. UAH 52 and S. viridosporus T7 A. From the results obtained it can be concluded that PY/GC/MS is a suitable technique to establish differences in the composition of the main product of solubilization of lignocellulose by different Streptomyces strains. Nevertheless for chemical characterization of this polymer, it is important to select the method to be used to extract the APPL from degraded residues in order to prevent chemical modifications of the substrate as occurred when NaOH was used which make difficult to attribute the changes observed to the microbial activity.

Acid-precipitable polymeric lignin from hardwood lignocellulose: production by a Streptomyces sp.

A Streptomyces sp. isolate, from decayed wood shavings, solubilized lignocellulose (LC) and lignin of Pinus radiata, producing about 50 mg acid-precipitable polymeric lignin per g LC. The product was poor in protein and carbohydrates and contained mainly vanillin, guaicol, vanillic and ferulic acids. Hardwood LC is thus suitable for producing APPL as a phenolic chemical feedstock.

Differently-induced extracellular phenol oxidases from Pleurotus ostreatus

Pleurotus ostreatus mycelium was grown in the presence of tobacco and wheat straw juices, ground wheat straw and acid-precipitable polymeric lignin with the aim of increasing the production of extracellular enzymes potentially implicated in lignin degradation. The highest production of diphenol oxidases was obtained when culture broths were supplemented with solid straw or tobacco juice; cellulase and veratryl alcohol oxidase were produced in detectable amounts in the presence of solid straw and in the absence of any inducer, respectively. No lignin peroxidase was found under any growth condition. Chromatographic fractionation of extracellular proteins and kinetic analysis of partially purified fractions showed that, in the absence of inducers, o-, p- and m-diphenol oxidases could be detected in column eluates, whereas all the inducers tested increased selectively the production of o-diphenol oxidases.

The effects of various nutrients on extracellular peroxidases and acid-precipitable polymeric lignin production byStreptomyces chromofuscus A2 andS. viridosporus T7A

The relationships between growth, medium pH, assimilation of glucose and amino acids, presence or absence of lignocellulose in the medium, lignin solubilization, and the appearance of extracellular peroxidase activity were compared for two lignin-solubilizing actinomycetes, Streptomyces chromofuscus A2 and S. viridosporus T7A. In a mineral salt medium containing yeast extract and three amino acids S. chromofuscus A2 grew faster than S. viridosporus T7A. When d-glucose was added to this medium, it was used in preference to the amino acids, the assimilation of which was delayed. Extracellular peroxidase activity peaked during the stationary phase, and glucose supplementation delayed peroxidase production. The eventual peak in peroxidase activity was higher in glucose-containing medium than in medium without glucose. Supplementation of the medium with lignocellulose did not affect either the level or time of appearance of extracellular peroxidase. However, lignin solubilization in lignocellulose-supplemented medium correlated positively with peroxidase activity: both increased after the cells entered the stationary phase. Supplementation of lignocellulose-containing medium with glucose delayed peroxidase production and lignin solubilization until the glucose had been assimilated. With S. viridosporus T7A, addition of d-glucose to the standard medium affected amino acid assimilation differently from S. chromofuscus A2. Glucose was consumed concomitantly with the amino acids. In the medium supplemented with lignocellulose, peroxidase activity and lignin solubilization correlated as they did for S. chromofuscus A2. A correlation of unknown significance was observed between the peroxidase activities of both strains and increasing medium pH. S. chromofuscus A2 produced more peroxidase and solubilized more lignin from lignocellulose than did S. viridosporus T7A. Overall, these findings show that extracellular peroxidases of both Streptomyces ssp. appear extracellularly primarily after cells cease growing and nutrients have been depleted from the medium. Also, increasing extracellular peroxidase activity and rates of lignin solubilization in both organisms are correlated and subject to glucose repression. These results point to the involvement of stationary-phase active peroxidases in the Streptomyces-catalyzed solubilization of lignin.

Lignin-solubilizing ability of actinomycetes isolated from termite (Termitidae) gut

The lignocellulose-degrading abilities of 11 novel actinomycete strains isolated from termite gut were determined and compared with that of the well-characterized actinomycete, Streptomyces viridosporus T7A. Lignocellulose bioconversion was followed by (i) monitoring the degradation of [14C]lignin- and [14C]cellulose-labeled phloem of Abies concolor to 14CO2 and 14C-labeled water-soluble products, (ii) determining lignocellulose, lignin, and carbohydrate losses resulting from growth on a lignocellulose substrate prepared from corn stalks (Zea mays), and (iii) quantifying production of a water-soluble lignin degradation intermediate (acid-precipitable polymeric lignin). The actinomycetes were all Streptomyces strains and could be placed into three groups, including a group of five strains that appear superior to S. viridosporus T7A in lignocellulose-degrading ability, three strains of approximately equal ability, and three strains of lesser ability. Strain A2 was clearly the superior and most effective lignocellulose decomposer of those tested. Of the assays used, total lignocellulose weight loss was most useful in determining overall bioconversion ability but not in identifying the best lignin-solubilizing strains. A screening procedure based on 14CO2 evolution from [14C-lignin]lignocellulose combined with measurement of acid-precipitable polymeric lignin yield was the most effective in identifying lignin-solubilizing strains. For the termite gut strains, the pH of the medium showed no increase after 3 weeks of growth on lignocellulose. This is markedly different from the pattern observed with S. viridosporus T7A, which raises the medium pH considerably. Production of extracellular peroxidases by the 11 strains and S. viridosporus T7A was followed for 5 days in liquid cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Selection of mutant Streptomyces strains with enhanced ability for acid precipitable polymeric lignin production

AbstractStreptomyces viridosporus T7A spores were subjected to ultraviolet (UV) light mutagensis. Two mutant strains S. viridosporus T7A‐40 and T7A‐198 were isolated. These strains were grown on 5 g of sugar cane baggase lignocellulose and produced 36.0 mg and 41.4 mg of acid precipitable polymeric lignin (APPL) respectively as compared to 25.1 mg produced by the wild type after 8 weeks incubation at 37 °C. The quantities of APPL produced by the mutants represent 43.7% and 65.3% more APPLs than those produced by the wild type. Apart from the increase in APPL production, the observable effects of the ultraviolet light included; variation in spore pigmentation, induction of a brown diffusible pigment, production of hydrogen sulfide, loss of thermotolerance, lower cellulase activity and loss of inositol utilizing ability.