Zygomycetes-based biorefinery: present status and future prospects.

Fungi of the phylum Zygomycetes fulfil all requirements for being utilized as core catalysts in biorefineries, and would be useful in creating new sustainable products. Apart from the extended use of Zygomycetes in preparing fermented foods, industrial metabolites such as lactic acid, fumaric acid, and ethanol are produced from a vast array of feedstocks with the aid of Zygomycetes. These fungi produce enzymes that facilitate their assimilation of various complex substrates, e.g., starch, cellulose, phytic acid, and proteins, which is relevant from an industrial point of view. The enzymes produced are capable of catalyzing various reactions involved in biodiesel production, preparation of corticosteroid drugs, etc. Biomass produced with the aid of Zygomycetes consists of proteins with superior amino acid composition, but also lipids and chitosan. The biomass is presently being tested for animal feed purposes, such as fish feed, as well as for lipid extraction and chitosan production. Complete or partial employment of Zygomycetes in biorefining procedures is consequently attractive, and is expected to be implemented within a near future.

Temperature shifts for extraction and purification of zygomycetes chitosan with dilute sulfuric acid.

The temperature-dependent hydrolysis and solubility of chitosan in sulfuric acid solutions offer the possibility for chitosan extraction from zygomycetes mycelia and separation from other cellular ingredients with high purity and high recovery. In this study, Rhizomucor pusillus biomass was initially extracted with 0.5 M NaOH at 120 °C for 20 min, leaving an alkali insoluble material (AIM) rich in chitosan. Then, the AIM was subjected to two steps treatment with 72 mM sulfuric acid at (i) room temperature for 10 min followed by (ii) 120 °C for 45 min. During the first step, phosphate of the AIM was released into the acid solution and separated from the chitosan-rich residue by centrifugation. In the second step, the residual AIM was re-suspended in fresh 72 mM sulfuric acid, heated at 120 °C and hot filtered, whereby chitosan was extracted and separated from the hot alkali and acid insoluble material (HAAIM). The chitosan was recovered from the acid solution by precipitation at lowered temperature and raised pH to 8-10. The treatment resulted in 0.34 g chitosan and 0.16 g HAAIM from each gram AIM. At the start, the AIM contained at least 17% phosphate, whereas after the purification, the corresponding phosphate content of the obtained chitosan was just 1%. The purity of this chitosan was higher than 83%. The AIM subjected directly to the treatment with hot sulfuric acid (at 120 °C for 45 min) resulted in a chitosan with a phosphate impurity of 18.5%.

Effects of different growth forms of Mucor indicus on cultivation on dilute-acid lignocellulosic hydrolyzate, inhibitor tolerance, and cell wall composition.

The dimorphic fungus Mucor indicus was grown in different forms classified as purely filamentous, mostly filamentous, mostly yeast-like and purely yeast-like, and the relationship between morphology and metabolite production, inhibitor tolerance and the cell wall composition was investigated. Low concentrations of spores in the inoculum with subsequent aeration promoted filamentous growth, whereas higher spore concentrations and anaerobic conditions promoted yeast-like growth. Ethanol was the main metabolite with glycerol next under all conditions tested. The yields of ethanol from glucose were between 0.39 and 0.42 g g(-1) with productivities of 3.2-5.0 g l(-1) h(-1). The ethanol productivity of mostly filamentous cells was increased from 3.9 to 5.0 g l(-1) h(-1) by the presence of oxygen, whereas aeration of purely yeast-like cells showed no such effect. All growth forms were able to tolerate 4.6 g l(-1) furfural and 10 g l(-1) acetic acid and assimilate the sugars, although with different consumption rates. The cell wall content of the fungus measured as alkali insoluble materials (AIM) of the purely yeast-like cells was 26% of the biomass, compared to 8% of the pure filaments. However, the chitosan concentration of the filaments was 29% of the AIM, compared to 6% of the yeast-like cells.

Determination of glucosamine and N-acetyl glucosamine in fungal cell walls.

A new method was developed to determine glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) in materials containing chitin and chitosan, such as fungal cell walls. It is based on two steps of hydrolysis with (i) concentrated sulfuric acid at low temperature and (ii) dilute sulfuric acid at high temperature, followed by one-step degradation with nitrous acid. In this process, chitin and chitosan are converted into anhydromannose and acetic acid. Anhydromannose represents the sum of GlcN and GlcNAc, whereas acetic acid is a marker for GlcNAc only. The method showed recovery of 90.1% of chitin and 85.7-92.4% of chitosan from commercial preparations. Furthermore, alkali insoluble material (AIM) from biomass of three strains of zygomycetes, Rhizopus oryzae, Mucor indicus, and Rhizomucor pusillus, was analyzed by this method. The glucosamine contents of AIM from R. oryzae and M. indicus were almost constant (41.7 +/- 2.2% and 42.0 +/- 1.7%, respectively), while in R. pusillus, it decreased from 40.0 to 30.0% during cultivation from 1 to 6 days. The GlcNAc content of AIM from R. oryzae and R. pusillus increased from 24.9 to 31.0% and from 36.3 to 50.8%, respectively, in 6 days, while it remained almost constant during the cultivation of M. indicus (23.5 +/- 0.8%).

Extraction and precipitation of chitosan from cell wall of zygomycetes fungi by dilute sulfuric acid.

A new method was developed in this work for extraction of chitosan from the zygomycetes cell wall. It is based on the temperature-dependent solubility of chitosan in dilute sulfuric acid. Chitin is soluble in neither cold nor hot dilute sulfuric acid. Similarly chitosan is not soluble at room temperature but is dissolved in 1% H 2SO 4 at 121 degrees C within 20 min. The new method was developed to measure the chitosan content of the biomass and cell wall. The procedures were investigated by measuring phosphate, protein, ash, glucuronic acid, and degree of acetylation. The cell wall derivatives of fungus Rhizomucor pusillus were then examined by this new method. The results indicated 8% of the biomass as chitosan. After treatment with NaOH, the alkali-insoluble material (AIM) contained 45.3% chitosan. Treatment of AIM with acetic acid resulted in 16.5% acetic-acid-soluble material (AcSM) and 79.0% alkali- and acid-insoluble material (AAIM). AcSM is usually cited as pure chitosan, but the new method shows major impurities by, for example, phosphate. Furthermore, AAIM is usually considered to be the chitosan-free fraction, whereas the new method shows more than 76% of the chitosan present in AIM is found in AAIM. It might indicate the inability of acetic acid to separate chitosan from the cell wall.

Fed-batch cultivation of Mucor indicus in dilute-acid lignocellulosic hydrolyzate for ethanol production.

Mucor indicus fermented dilute-acid lignocellulosic hydrolyzates to ethanol in fed-batch cultivation with complete hexose utilization and partial uptake of xylose. The fungus was tolerant to the inhibitors present in the hydrolyzates. It grew in media containing furfural (1 g/l), hydroxymethylfurfural (1 g/l), vanillin (1 g/l), or acetic acid (7 g/l), but did not germinate directly in the hydrolyzate. However, with fed-batch methodology, after initial growth of M. indicus in 500 ml enzymatic wheat hydrolyzate, lignocellulosic hydrolyzate was fermented with feeding rates 55 and 100 ml/h. The fungus consumed more than 46% of the initial xylose, while less than half of this xylose was excreted in the form of xylitol. The ethanol yield was 0.43 g/g total consumed sugar, and reached the maximum concentration of 19.6 g ethanol/l at the end of feeding phase. Filamentous growth, which is regarded as the main obstacle to large-scale cultivation of M. indicus, was avoided in the fed-batch experiments.

Ethanol production from hexoses, pentoses, and dilute-acid hydrolyzate by Mucor indicus.

Consumption of hexoses and pentoses and production of ethanol by Mucor indicus were investigated in both synthetic media and dilute-acid hydrolyzates. The fungus was able to grow in a poor medium containing only carbon, nitrogen, phosphate, potassium, and magnesium sources. However, the cultivation took more than a week and the ethanol yield was only 0.2 gg(-1). Enrichment of the medium by addition of trace metals, particularly zinc and yeast extract, improved the growth rate and yield, such that the cultivation was completed in less than 24 h and the ethanol and biomass yields were increased to 0.40 and 0.20 gg(-1), respectively. The fungus was able to assimilate glucose, galactose, mannose, and xylose, and produced ethanol with yields of 0.40, 0.34, 0.39, and 0.18 gg(-1), respectively. However, arabinose was poorly consumed and no formation of ethanol was detected. Glycerol was the major by-product in the cultivation on the hexoses, while formation of glycerol and xylitol were detected in the cultivation of the fungus on xylose. The fungus was able to take up the sugars present in dilute-acid hydrolyzate as well as the inhibitors, acetic acid, furfural, and hydroxymethyl furfural. M. indicus was able to grow under anaerobic conditions when glucose was the sole carbon source, but not on xylose or the hydrolyzate. The yield of ethanol in anaerobic cultivation on glucose was 0.46 g g(-1).

Production of mycelium biomass and ethanol from paper pulp sulfite liquor by Rhizopus oryzae.

The cultivation conditions for Rhizopus oryzae grown in synthetic medium and paper pulp spent sulfite liquor (SSL) were investigated to achieve high biomass and ethanol yields using shake flasks and bioreactors. The fungus assimilated the hexoses glucose, mannose and galactose, and the pentoses xylose and arabinose as well as acetic acid which are present in SSL. The assimilation of hexoses was faster than pentoses during cultivation in a synthetic medium. However, all sugars were assimilated concomitantly during growth in SSL supplemented with ammonium, magnesium, calcium, phosphate, sulfate and trace amounts of some other metal ions (SSL-S). The medium composition had an important influence on biomass yield. The highest biomass yields, viz. 0.18 and 0.43 g biomass/g sugar were obtained, when the cells were cultivated in shake flasks with a synthetic medium containing glucose as carbon and energy source and SSL-S, respectively. The corresponding yields in a bioreactor with more efficient aeration were 0.22 and 0.55 g/g. In addition to the biomass, ethanol, lactic acid, and glycerol were important extracellular metabolites of the cultivation with maximum yields of 0.37, 0.30 and 0.09 g/g, respectively. When the source of sugars in the medium was exhausted, the fungus consumed the metabolites produced, such that the liquid medium was depleted of potential oxidizable nutrients. In general, there was a direct competition between lactic acid and ethanol among the metabolites. Poor medium compositions and cultivation conditions resulted in higher yields of lactic acid, whereas the ethanol and biomass yields were higher in rich media. SSL-S supported good growth of mycelium and a high ethanol yield.

Hydrolysis of the soft amphiphilic antimicrobial agent tetradecyl betainate is retarded after binding to and killing Salmonella typhimurium.

Hydrolysis of tetradecyl betainate (B14), a fast-acting microbicidal amphiphilic quaternary ammonium compound (QAC) being an ester of betaine and tetradecanol, occurred after binding to Salmonella typhimurium, resulting in release of the water-soluble betaine portion and retention of the lipophilic tetradecanol. The rate of the hydrolysis was significant but retarded in comparison to B14 in solution. As in free solution, the hydrolysis of substance bound to S. typhimurium was increased in an alkaline environment and by heat. At pH 6.0 and 20 degrees C the hydrolysis of bound ester was about 10% after 180 min, whereas at pH 9.0 and 50 degrees C it was about 50% after 60 min. These results are consistent with a model where amphiphilic QACs are inserted into the bacterial outer membrane (OM) with the quaternary ammonium head group facing outwards and the lipophilic portion, including the ester bond, being in the membrane lipid environment enough for retarding the hydrolysis. However, calculation of the mean concentration of B14 in the bacteria at MBC99 (minimum bactericidal concentration required to kill 99% of cells) showed a 7000-8000 times greater concentration than in the medium. At this concentration, when most B14 is considered to be bound to the OM, the available surface area for each molecule was only 2 A2. This is only 6-7% of that required for close packing of the quaternary ammonium head group (30 A2), indicating that a three-dimensional, presumably continuous arrangement was formed. Since B14 is hydrolysed after its binding to bacteria with microbicidal effect, it may be used under conditions where stable QACs might be harmful to the close or the common environment.