Purification of the extracellular pectinolytic enzyme from the fungus Rhizopus oryzae NBRC 4707

The pectinolytic enzyme from the solid-state culture of Rhizopus oryzae NBRC 4707 was purified to homogeneity by column chromatography on CM-Toyopearl 650 M and hydroxylapatite. The molecular weight of the enzyme was estimated by SDS-polyacrylamide gel electrophoresis to be 31,000 and was reduced to 29,700 after treatment with endoglycosidase H. Maximal activity was observed near pH 4.5 at 45°C. The enzyme was shown to be endopolygalacturonase, as judged from the formation of oligogalacturonides as its reaction products. The addition of purified enzyme, as expected, enhanced the formation of lactic acid and ethanol in potato pulp grown with R. oryzae.     

Optimization of the Process for the Production of L(+)‐Lactic Acid from Cull Potato by Rhizopus oryzae

Cull potato is currently an under‐utilized biomass in the potato processing states of the USA. L(+)‐Lactic acid production by three Rhizopus strains and one homofermentative, facultative anaerobic Lactobacillus amylophilus strain was investigated using potatoes as the sole nutrient supply in the culture medium. Rhizopus oryzae NRRL 395 was chosen as the strain for further studies because it showed the highest lactate yield. The fermentation conditions for seed cultures were studied for three treatment structures using a completely randomized design. Optimum conditions for the seed culture were determined to be 2 % potato medium, 10⁴ spores/mL concentration, and 24 h of fermentation. Plackett‐Burman and central composite designs were used to screen and optimize the factors for lactic acid production. Substrate (potato) concentration, fermentation temperature, and shaking speed were found to be the most significant factors affecting both the yield and concentration of lactate. Optimum values for substrate concentration, fermentation temperature, and shaking speed were 10 %, 27 °C, and 170 rpm, respectively. Under these optimum conditions, the lactate concentration was predicted by the model to be 35.5 g/L, which was verified by the experimental data (33.3 g/L). The results indicate that cull potato can be an effective feedstock for R. ryzae NRRL 395 in the production of lactic acid.     

Co-production of lactic acid and chitin using a pelletized filamentous fungus Rhizopus oryzae cultured on cull potatoes and glucose

Aims: This paper developed a novel process for lactic acid and chitin co-production of the pelletized Rhzious oryzae NRRL 395 fermentation using underutilized cull potatoes and glucose as nutrient source. Methods and Results: Whole potato hydrolysate medium was first used to produce the highest pelletized biomass yield accompanying the highest chitin content in biomass. An enhanced lactic acid production then followed up using batch, repeated batch and fed batch culture with glucose as carbon source and mixture of ammonia and sodium hydroxide as neutralizer. The lactic acid productivity peaked at 2·8 and 3 g l⁻¹ h⁻¹ in repeated batch culture and batch culture, respectively. The fed batch culture had the highest lactate concentration of 140 g l⁻¹. Conclusions: Separation of the biomass cultivation and the lactic acid production is able to not only improve lactic acid production, but also enhance the chitin content. Cull potato hydrolysate used as a nutrient source for biomass cultivation can significantly increase both biomass yield and chitin content. Significance and Impact of the Study: The three-step process using pelletized R. oryzae fermentation innovatively integrates utilization of agricultural residues into the process of co-producing lactic acid and chitin, so as to improve the efficiency, revenues and cost of fungal lactic acid production.     

Production of lactic acid and ethanol by Rhizopus oryzae integrated with cassava pulp hydrolysis

Cassava pulp was hydrolyzed with acids or enzymes. A high glucose concentration (>100 g/L) was obtained from the hydrolysis with 1 N HCl at 121 °C, 15 min or with cellulase and amylases. While a high glucose yield (>0.85 g/g dry pulp) was obtained from the hydrolysis with HCl, enzymatic hydrolysis yielded only 0.4 g glucose/g dry pulp. These hydrolysates were used as the carbon source in fermentation by Rhizopus oryzae NRRL395. R. oryzae could not grow in media containing the hydrolysates treated with 1.5 N H₂SO₄ or 2 N H₃PO₄, but no significant growth inhibition was found with the hydrolysates from HCl (1 N) and enzyme treatments. Higher ethanol yield and productivity were observed from fermentation with the hydrolysates when compared with those from fermentation with glucose in which lactic acid was the main product. This was because the extra organic nitrogen in the hydrolysates promoted cell growth and ethanol production.     

Comparison of Sucrose-Hydrolyzing Enzymes Produced by Rhizopus oryzae and Amylomyces rouxii

Rhizopus oryzae produces lactic acid from glucose but not efficiently from sucrose, while Amylomyces rouxii, a species closely related to R. oryzae, ferments these sugars equally. The properties of two sucrose-hydrolyzing enzymes purified from culture filtrates of R. oryzae NBRC 4785 and A. rouxii CBS 438.76 were compared to assess lactic acid fermentation by the two fungi. The substrate specificity of the enzymes showed that the enzymes from strains NBRC 4785 and CBS 438.76 are to be classified as glucoamylase and invertase respectively. The entity of the enzyme from strain NBRC 4785 might be a glucoamylase, because eight residues of the N-terminal amino acid sequence coincided with those of the deduced protein from the amyB gene of R. oryzae. The enzyme from NBRC 4785 was more unstable than that from strain CBS 438.76 under conditions of lower pH and higher temperature. These observations mean that the culture conditions of R. oryzae for lactic acid production from sucrose should be strictly controlled to prevent inactivation of the glucoamylase hydrolyzing sucrose.     

Kinetics of direct fermentation of agricultural commodities to L(+) lactic acid byRhizopus oryzae

Summary The kinetics of fermenting barley, cassava, corn, oats, and rice directly to L(+) lactic acid byRhizopus oryzae NRRL 395 were determined. The rates of carbohydrate consumption and L(+) lactic acid production were found to be influenced by the type of substrate, the substrate concentration, the fermentation temperature, and the presence of a neutralizing agent.     

Direct fermentation of potato starch in wastewater to lactic acid by<Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

The fungal species ofRhizopus oryzae 2062 has the capacity to carry out a single stage fermentation process for lactic acid production from potato starch wastewater. Starch hydrolysis, reducing sugar accumulation, biomass formation, and lactic acid production were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/L at pH 6.0 and 30°C was favourable for starch fermentation, resulting in a lactic acid yield of 78.3%–85.5% associated with 1.5–2.0 g/L fungal biomass produced in 36 h of fermentation.     

Mass production of spores of lactic acid‐producing Rhizopus oryzae NBRC 5384 on agar plate

Mass production of sporangiospores (spores) of Rhizopus oryzae NBRC 5384 (identical to NRRL 395 and ATCC 9363) on potato‐dextrose‐agar medium was studied aiming at starting its L (+)‐lactic acid fermentation directly from spore inoculation. Various parameters including harvest time, sowed spore density, size of agar plate, height of air space, and incubation mode of plate (agar‐on‐bottom or agar‐on‐top) were studied. Ordinarily used shallow Petri dishes were found out to be unsuitable for the full growth of R. oryzae sporangiophores. In a very wide range of the sowed spore density, the smaller it was, the greater the number of the harvested spores was. It was also interesting to find out that R. oryzae grown downward vertically with a deep air space in an agar‐on‐top mode gave larger amount of spores than in an agar‐on‐bottom mode at 30°C for 7‐day cultivation. Scale‐up of the agar plate culture from 26.4 to 292 cm² was studied, resulting in the proportional relationship between the number of the harvested spores/plate and the plate area in the deep Petri dishes. The number of plates of 50 cm in diameter needed for 100 m³ industrial submerged fermentation started directly from 2 × 10⁵ spores/mL inoculum size was estimated as about 6, from which it was inferred that such a fermentation would be feasible. Designing a 50 cm plate and a method of spreading and collecting the spores were suggested. Bioprocess technological significance of the “full‐scale industrial submerged fermentation started directly from spore inoculation omitting pre‐culture” has been discussed. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:876–881, 2013     

Production of lactic acid and fungal biomass by Rhizopus fungi from food processing waste streams

This study proposed a novel waste utilization bioprocess for production of lactic acid and fungal biomass from waste streams by fungal species of Rhizopus arrhizus 36017 and R. oryzae 2062. The lactic acid and fungal biomass were produced in a single-stage simultaneous saccharification and fermentation process using potato, corn, wheat and pineapple waste streams as production media. R. arrhizus 36017 gave a high lactic acid yield up to 0.94–0.97 g/g of starch or sugars associated with 4–5 g/l of fungal biomass produced, while 17–19 g/l fungal biomass with a lactic acid yield of 0.65–0.76 g/g was produced by the R. oryzae 2062 in 36–48 h fermentation. Supplementation of 2 g/l of ammonium sulfate, yeast extract and peptone stimulated an increase in 8–15% lactic acid yield and 10–20% fungal biomass.

     

A flax-retting endopolygalacturonase-encoding gene from Rhizopus oryzae

A polygalacturonase from the filamentous fungus Rhizopus oryzae strain sb (NRRL 29086), previously shown to be effective in the retting of flax fibers, was shown by the analysis of its reaction products on polygalacturonic acid to be an endo-type. By zymogram analysis, the enzyme in the crude culture filtrate appeared as two active species of 37 and 40 kD. The endopolygalacturonase-encoding gene was cloned in Escherichia coli and its translated 383-amino acid sequence found to be identical to that of a presumed exopolygalacturonase found in R. oryzae strain YM9901 and 96% identical to a hypothetical protein (RO3G_04731.1) in the sequenced genome of R. oryzae strain 99–880. Phylogenetic analysis revealed the presence of an unique cluster of Rhizopus polygalacturonase sequences that are separate from other fungal polygalacturonases. Conservation of 12 cysteines appears to be a special feature of this family of Rhizopus polygalacturonase sequences. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production and characterization of chitosan obtained from <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> grown on potato chip processing waste

Potato chip processing waste of trimmed potato, potato peel and substandard (low-quality) potato chips, obtained from a potato chip processing plant, were used as substrates for chitosan production from Rhizopus oryzae. It was cultured on each waste product at 30 ± 2°C and 70% moisture content for 21 days. Fermented potato peel had the highest yield after 5 days of fermentation. The cultivation condition of chitosan obtained from R. oryzae was optimum for a peel size of less than 6 mesh, 70% moisture content and a pH of 5. Furthermore, the best extraction condition was using 46% sodium hydroxide at 46°C for 13 h followed by 2% acetic acid at 95°C for 8 h. The maximum chitosan yield obtained by these conditions was 10.8 g/kg substrate. Fungal chitosan properties were found to be 86–90% degree of deacetylation, molecular weight of 80–128 kDa and viscosity of 3.1–6.1 mPa s. Therefore, potato peel could be applied as a low cost substrate for chitosan production from R. oryzae.     

The fermentation of cell wall substances in grass silage and in potato pulp

Summary The amount of acid formed in grass silage was greater than could have been formed from the soluble sugars present, even when only a lactic fermentation took place. This seemed to point to fermentation of cell wall substances by lactic acid bacteria. Lactic acid fermentation in potato pulp always takes place with cell wall substances as substrates, as sugars are absent. It was found that galactose, probably occurring as galactan, and also some pectic acid were fermented in potato pulp. Some lactobacilli were isolated from potato pulp; streptobacteria which could ferment galactan but no pectic or galacturonic acid, and betabacteria which could ferment galacturonic acid but no galactan or pectic acid. A number of homofermentative lactobacilli were all found to belong to the speciesStreptobacterium casei. It was shown that a strain of this species could ferment galactan in potato pulp sterilised previously with ethylene oxide. Part of this work was carried out at the Netherlands Institute for Dairy Research, Ede, Netherlands.     

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30°C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87–0.97 g/g starch associated with 1.5–2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.     

Increased Levels of Policosanol and Very Long-Chain Fatty Acids in Potato Pulp Fermented with Rhizopus oryzae

Significant amounts of policosanol and very long-chain fatty acids (VLFAs) ranging in carbon length from 22 to 30 were found in the lipophilic fraction obtained from potato pulp fermented with Rhizopus oryzae. It is believed that these compounds would have originally been present as suberin-related compounds, but not as wax, in the periderm of potato tubers and concentrated into potato pulp during the process of starch production. Moreover, the policosanol and VLFAs extracted from potato pulp with organic solvents were found to have increased after fermentation.     

Complete genome sequence of Lactobacillus buchneri NRRL B-30929, a novel strain from a commercial ethanol plant

Lactobacillus buchneri strain NRRL B-30929 was a contaminant obtained from a commercial ethanol fermentation. This facultative anaerobe is unique because of its rapid growth on xylose and simultaneous fermentation of xylose and glucose. The strain utilizes a broad range of carbohydrate substrates and possesses a high tolerance to ethanol and other stresses, making it an attractive candidate for bioconversion of biomass substrates to various bioproducts. The genome sequence of NRRL B-30929 will provide insight into the unique properties of this lactic acid bacterium.     

Efficient harvesting of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 with filamentous fungal pellets

Cyanobacteria play a major role as direct producers of biofuels, such as ethanol and butanol, with the aid of genetic engineering. However, development of a new harvesting-technology is essential to achieve economic viability of biofuel production from cyanobacteria. In this study, we demonstrated the feasibility of harvesting the unicellular cyanobacterium Synechocystis sp. PCC 6803 using pre-made filamentous fungal pellets and investigated key factors affecting efficiency of harvest, including fungal strain, pellet quantity (number of pellets), initial pH, and organic carbon source. Synechocystis sp. PCC 6803 cells attached to Aspergillus oryzae pellets, indicating that this fungal pellet had a desirable harvesting effect, while Rhizopus oryzae pellets had no effect on harvesting. Increasing pellet quantity and adding organic carbon sources, such as glucose and xylose, improved the harvesting efficiency of Aspergillus oryzae pellet; efficiency was not affected by the initial pH.     

Effects of promoters on the enhancement of pectin methyl esterase expression inAspergillus niger

Summary A pectin methylesterase-encoding gene (pmeA)_has been cloned and transformed intoA. niger wild-type NRRL3. Transformants produced 20-fold more PME than the host strain. For studying the effects of different promoters on thepmeA expression two novel plasmids were constructed, in which thepmeA promoter was replaced by efficient promoters such as theA. nidulans glyceraldehyde-3-phosphate dehydrogenase (pK45) or theA. oryzae -amylase (pK61) promoter. The highest level of PME expression was achieved with theA. oryzae -amylase promoter, reaching a 200-fold increase compared to the production by the host strain.     

Survival strategy of the salt-tolerant lactic acid bacterium,Tetragenococcus halophilus,to counteract koji mold,Aspergillus oryzae,in soy sauce brewing

In soy sauce brewing, the results of the fermentation of lactic acid greatly affect the quality of soy sauce. The soy sauce moromi produced with Aspergillus oryzae RIB40 allows the growth of Tetragenococcus halophilus NBRC 12172 but not T. halophilus D10. We isolated and identified heptelidic acid (HA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), produced by A. oryzae RIB40 as the growth inhibitor of the salt-tolerant lactic acid bacteria. The growth inhibition of T. halophilus D10 by HA was suggested to be associated with the direct inhibition of GAPDH activity under high salt environment. The difference in the susceptibility to HA among various strains of T. halophilus was caused by the mutations in the gene encoding GAPDH.     

Comparative evaluation of Aspergillus niger strains for endogenous pectin-depolymerization capacity and suitability for d-galacturonic acid production

Pectinaceous agricultural residues rich in d-galacturonic acid (d-GalA), such as sugar beet pulp, are considered as promising feedstocks for waste-to-value conversions. Aspergillus niger is known for its strong pectinolytic activity. However, while specialized strains for production of citric acid or proteins are well characterized, this is not the case for the production of pectinases. We, therefore, systematically compared the pectinolytic capabilities of six A. niger strains (ATCC 1015, ATCC 11414, NRRL 3122, CBS 513.88, NRRL 3, and N402) using controlled batch cultivations in stirred-tank bioreactors. A. niger ATCC 11414 showed the highest polygalacturonase activity, specific protein secretion, and a suitable morphology. Furthermore, d-GalA release from sugar beet pulp was 75% higher compared to the standard lab strain A. niger N402. Our study, therefore, presents a robust initial strain selection to guide future process improvement of d-GalA production from agricultural residues and identifies a high-performance base strain for further genetic optimizations.

     

Biotechnological production of lactic acid integrated with fishmeal wastewater treatment by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

Fishmeal wastewater, a seafood processing waste, was utilized for production of lactic acid and fungal biomass by Rhizopus oryzae AS 3.254 with the addition of sugars. The 30 g/l exogenous glucose in fishmeal wastewater was superior to starch in view of productivities of lactic acid and fungal biomass, and COD reduction. Fishmeal wastewater can be a replacement for peptone which was the most suitable nitrogen source for lactic acid production among the tested organic or inorganic nitrogen sources. Exogenous NaCl (12 g/l) completely inhibited the production of lactic acid and fungal growth. In the medium of COD 5,000 mg/l fishmeal wastewater with the addition of 30 g/l glucose, the maximum productivity of lactic acid was 0.723 g/l h corresponding to productivity of fungal biomass 0.0925 g/l h, COD reduction 84.9% and total nitrogen removal 50.3% at a fermentation time of 30 h.