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.

Spent sulphite liquor, the major byproduct from the sulphite pulp production process, was diluted to 50% and used for production of an edible zygomycete Rhizopus sp. The focus was on production, yield, and composition of the fungal biomass composition. The fungus grew well at 20 to 40°C, but 32°C was found to be preferable compared to 20 and 40°C in terms of biomass production and yield (maximum of 0.16 g/g sugars), protein content (0.50-0.60 g/g), alkali-insoluble material (AIM) (ca 0.15 g/g), and glucosamine content (up to 0.30 g/g of AIM). During cultivation in a pilot airlift bioreactor, the yield increased as aeration was raised from 0.15 to 1.0 vvm, indicating a high demand for oxygen. After cultivation at 1.0 vvm for 84 h, high yield and production of biomass (up to 0.34 g/g sugars), protein (0.30-0.50 g/g), lipids (0.02-0.07 g/g), AIM (0.16-0.28 g/g), and glucosamine (0.22-0.32 g/g AIM) were obtained. The fungal biomass produced from spent sulphite liquor is presently being tested as a replacement for fishmeal in feed for fish aquaculture and seems to be a potential source of nutrients and for production of glucosamine.

The capability of two zygomycetes strains. Mucor indicus and an isolate from tempeh (Rhizopus sp.), to grow on orange peel hydrolysate and their tolerance to its antimicrobial activity, was investigated. Both fungi, in particular M. indicus, tolerated up to 2% D-limonene in semi-synthetic media during cultivation in shake flasks, under aerobic as well as anaerobic conditions. The tolerance of M. indicus was also tested in a bioreactor, giving rise to varying results in the presence of 2% limonene. Furthermore, both strains were capable of consuming galacturonic acid, the main monomer of pectin, under aerobic conditions when no other carbon source was present. The orange peel hydrolysate was based on 12% (dry w/v) orange peels, containing D-limonene at a concentration of 0.6% (v/v), which no other microorganism has been reported to be able to ferment. However, the hydrolysate was utilised by M. indicus under aerobic conditions, resulting in production of 410 and 400 mg ethanol/g hexoses and 57 and 75 mg fungal biomass/g sugars from cultivations in shake flasks and a bioreactor, respectively. Rhizopus sp., however, was slow to germinate aerobically, and neither of the zygomycetes was able to consistently germinate in orange peel hydrolysate. under anaerobic conditions. The zygomycetes strains used in the present study demonstrated a relatively high resistance to the antimicrobial compounds present in orange peel hydrolysate, and they were capable of producing ethanol and biomass in the presence of limonene, particularly when cultivated with air supply. (C) 2012 Elsevier Ltd. All rights reserved.

The metabolite profiles from muscles and livers of Atlantic salmon were investigated using high resolution nuclear magnetic resonance (HR-NMR) spectroscopy of aqueous extracts and magic angle spinning (MAS) NMR spectroscopy of intact tissues. Comparison of the data showed that most small metabolites present in the aqueous extracts were also identified in the Carr–Purcell–Meiboom–Gill (CPMG) MAS NMR spectra of the intact tissues. Not only the total omega-3 fatty acid content, but also the EPA and DHA content, in the muscle and liver tissues could be calculated directly from the diffusion-edited MAS NMR spectra without the need for lipophilic extraction. The effect of replacing a normal fish meal during a period of 3months with a diet containing 20% zygomycete was also investigated by multivariate analysis of the NMR spectra. Principal component analysis (PCA) was used to study the small metabolites distribution in the aqueous extracts of liver and muscles, and revealed that the feed containing zygomycete could have an influence on the metabolites profiles of juvenile Atlantic salmon.

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%.

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 0.42gg−1 with productivities of 3.2–5.0gl−1h−1. The ethanol productivity of mostly filamentous cells was increased from 3.9 to 5.0gl−1h−1 by the presence of oxygen, whereas aeration of purely yeast-like cells showed no such effect. All growth forms were able to tolerate 4.6gl−1 furfural and 10gl−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.

The dimorphic filamentous fungus Mucor indicus was used as a biofilter and fermenting microorganism in a bioreactor for continuous cultivation of dilute-acid lignocellulosic hydrolyzates up to dilution rate (D) 0.3h−1. The hydrolyzate was so toxic that the fungus could not grow in traditional stirred-tank bioreactors even at D 0.1h−1. On the other hand, the fungus could be manipulated to grow as a mixture of short dispersed filaments and yeast-like cells. This morphology was suitable to create a biological filter, when attached to a specific screen located across the outlet stream. This filter was fully permeable to the liquid, but not the biomass inside the bioreactor. While the biomass concentration in the outflow was between 0.51 and 3.43g/l at dilution rates 0.3–0.1h−1, the biomass concentration inside the bioreactor was held at 8.34–9.91g/l. The cells consumed 95.7%, 94.3%, and 75.3% of hexoses and 64.5%, 25.9%, and 7.6% of xylose present in the hydrolyzate at D 0.1, 0.2, and 0.3h−1, respectively. The ethanol yields at these dilution rates were 0.37, 0.45, and 0.46g/g consumed sugars, respectively. The only important by-products were glycerol and xylitol, which at D 0.2h−1 yielded 0.036 and 0.022g/g consumed sugars, respectively.

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%).

The fungus Mucor indicus is able to produce ethanol from xylose as well as dilute-acid lignocellulosic hydrolyzates. The fungus completely assimilated 10 g/L xylose as the sole carbon and energy source within 32 to 65 h at an aeration rate of 0.1 to 1.0 vvm. The highest ethanol yield was 0.16 g/g at 0.1 vvm. Xylitol was formed intermediately with a maximum yield of 0.22 g/g at 0.5 vvm, but disappeared towards the end of experiments. During cultivation in a mixture of xylose and glucose, the fungus did not assimilate xylose as long as glucose was present in the medium. The anaerobic cultivation of the fungus in the hydrolyzate containing 20% xylose and 80% hexoses resulted in no assimilation of xylose but complete consumption of the hexoses in less than 15 h. The ethanol yield was 0.44 g/g. However, the xylose in the hydrolyzate was consumed when the media was aerated at 0.067 to 0.333 vvm. The best ethanol yield was 0.44 g/g at 0.067 vvm. The results of this study suggest that M. indicus hydrolyzate can be first fermented anaerobically for hexose assimilation and subsequently continued under oxygen-limited conditions for xylose fermentation.

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.

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.

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.

In searching for ethanol producing microorganisms also capable of fermenting pentoses, nine zygomycetes strains including three strains of Rhizopus oryzae, Mucor corticolous, M. hiemalis, M. indicus, Rhizomucor pusillus, R. miehei, and zygomycete IT were examined. Each strain was cultivated on glucose, xylose or dilute-acid hydrolyzate (DAH) as carbon sources, and the production of ethanol, lactic acid, glycerol, xylitol, and succinic acid were investigated. Great similarities but also conspicuous differences were seen between the species, to some extent linked to the genera. All strains were capable of growing on glucose or xylose as single carbon source. With the exception of the two Rhizomucor strains, all produced ethanol. All the strains produced glycerol as by-product, while Rhizopus and Rhizomucor but not Mucor produced lactic acid in significant amounts. All Mucor and Rhizopus strains and one strain of Rhizomucor produced xylitol in the xylose medium, but no xylitol was detected after growth on DAH. All Mucor and two R. oryzae strains were capable of growing on DAH. Two Mucor species, M. hiemalis and M. indicus showed greater ethanol production than the other strains. The ethanol yields by M. hiemalis on glucose, xylose, and DAH were 0.39, 0.18, and 0.44g/g, respectively, whereas the corresponding results for M. indicus were 0.39, 0.22, and 0.44g/g. The strains also rapidly consumed hydroxymethyl furfural present in DAH.

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.

The presence of diarrheic shellfish toxins (DST) has been the main obstacle to mussel cultivation in Sweden. Monitoring of DST concentrations in mussels by HPLC for 12 years has shown great geographical and seasonal differences. Furthermore, individual mussels on the same farming strip may differ by a factor as great as 16. DST levels are usually low in summer, rise in September, remain high in the winter and vanish after the start of the spring bloom. A part of the Bohuslan fjord system north of the island Orust, which is secluded from the open sea by shallow sills, regularly shows low DST concentrations. This basin is among the most eutrophicated parts of the Bohuslan coastline. Cultivation of mussels in this area has been calculated to reduce the eutrophication, and results of field-tests support this view. As a consequence, a strategy for sea-gardening is being developed in which the presence of nutrients with primary production in the sea and cultivation of mussels are planned to balance one another.