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In previous work, we proposed a novel modified one-step fermentation fed-batch strategy to efficiently generate L-lactic acid (L-LA) using Rhizopus oryzae. In this study, to further enhance efficiency of L-LA production through one-step fermentation in fed-batch cultures, we systematically investigated the initial peptone- and glucose-feeding approaches, including different initial peptone and glucose concentrations and maintained residual glucose levels. Based on the results of this study, culturing R. oryzae with initial peptone and glucose concentrations of 3.0 and 50.0 g/l, respectively, using a fed-batch strategy is an effective approach of producing L-LA through one-step fermentation. Changing the residual glucose had no obvious effect on the generation of L-LA. We determined the maximum LA production and productivity to be 162 g/l and 6.23 g/(l·h), respectively, during the acid production stage. Compared to our previous work, there was almost no change in L-LA production or yield; however, the productivity of L-LA increased by 14.3%.

Consolidated bioprocessing (CBP) is a potential breakthrough technology for reducing costs of biochemical production from lignocellulosic biomass. Production of cellulase enzymes, saccharification of lignocellulose, and conversion of the resulting sugars into a chemical of interest occur simultaneously within a single bioreactor. In this study, synthetic fungal consortia composed of the cellulolytic fungus Trichoderma reesei and the production specialist Rhizopus delemar demonstrated conversion of microcrystalline cellulose (MCC) and alkaline pre-treated corn stover (CS) to fumaric acid in a fully consolidated manner without addition of cellulase enzymes or expensive supplements such as yeast extract. A titer of 6.87 g/L of fumaric acid, representing 0.17 w/w yield, were produced from 40 g/L MCC with a productivity of 31.8 mg/L/hr. In addition, lactic acid was produced from MCC using a fungal consortium with Rhizopus oryzae as the production specialist. These results are proof-of-concept demonstration of engineering synthetic microbial consortia for CBP production of naturally occurring biomolecules., (© 2017 Wiley Periodicals, Inc.)

Lactic acid has a wide industrial application area and can be produced by fungal strains. However, excessive bulk growth form of fungi during the fermentations is a major problem, which limits the fermentation performance. Microparticles are excellent tools to prevent bulk fungal growth and provide homogenized fermentation broth to increase uniformity and the prediction performance of the models. Therefore, in this study, addition of aluminum oxide and talcum microparticles into fermentations was evaluated to enhance the production of lactic acid by Rhizopus oryzae. The results showed that the bulk fungal growth was prevented and the lactic acid concentration increased from 6.02 to 13.88 and 24.01 g/L, when 15 g/L of aluminum oxide or 10 g/L of talcum was used, respectively, in the shake-flask fermentations. Additionally, substrate concentration, pH, and agitation were optimized in the bioreactors using response surface methodology, and optimum values were determined as 126 g/L of glucose, 6.22 pH, and 387 rpm, respectively. Under these conditions, lactic acid production further increased to 75.1 ± 1.5 g/L with 10 g/L of talcum addition. Also, lactic acid production and glucose consumption in the batch fermentation were successfully modeled with modified Gompertz model and modified logistic model. RMSE and MAE values for lactic acid production were calculated as 2.279 and 1.498 for the modified Gompertz model; 3.6 and 4.056 for the modified logistic model. Additionally, modified logistic model predicted glucose consumption with -2.088 MAE and 2.868 RMSE, whereas these values were calculated as 2.035 and 3.946 for the modified Gompertz model.

The effects of pellet morphology, diameter, density, and interior structure on L-lactic acid fermentation by Rhizopus oryzae were characterized for different inoculum sizes and concentrations of peptone and CaCO3. Inoculum size was the most important factor determining pellet formation and diameter. The diameter decreased with increasing inoculum size, and larger pellets were observed for lower inoculum sizes. Peptone concentration had the greatest effect on pellet density, which increased with increasing peptone concentration. L-lactic acid production depended heavily on pellet density but not on pellet diameter. Low-density pellets formed easily under conditions of low peptone concentration and often had a relatively hollow structure, with a thin condensed layer surrounding the pellet and an extraordinarily loose biomass or hollow center. As expected, this structure greatly decreased production. The production of L-lactic acid increased until the density reached a certain level (50-60 kg/m(3)), in which the compact part was distributed homogeneously in the thick outer layer of the pellet and loose in the central layer. Homogeneously structured, denser pellets had limited mass transfer, causing a lower overall turnover rate. However, the interior structure remained nearly constant throughout all fermentation phases for pellets with the same density. CaCO3 concentration only had a slight influence on pellet diameter and density, probably because it increases spore germination and filamentous hypha extension. This work also provides a new analysis method to quantify the interior structure of pellets, thus giving insight into pellet structure and its relationship with productivity.

This study shows that Rhizopus oryzae is capable of directly utilizing cassava pulp alone to L-lactic acid in solid state fermentation (SSF). pH control at 6.0 helped prevent end product inhibition. Increasing lactate titer was observed at the higher initial moistened water due to the higher degree of substrate swelling and hydrolysis. With shaking, limited ethanol production but no change in lactate titer was observed. Rigorous shaking gave better oxygen transfer but presumably caused cell damage leading to substrate utilization through the biosynthesis route. Supplementing cassava pulp with nitrogen enhanced growth but not lactate production. Under the optimal conditions, R. oryzae converted the sole cassava pulp into lactic acid at the titer of 206.20 mg per g initial dry pulp. With the help of commercial cellulase and glucoamylase, the dramatically increasing lactate titer of 463.18 mg per g initial dry pulp was achieved via SSF.

Lactic acid, an anaerobic product of glycolysis, can be theoretically produced by synthetic route; however, it is commercially produced by homo-fermentative batch mode of operations. Factors affecting its production and strategies improving it are considered while devising an optimized protocol. Although a hetero-fermentative mode of production exists, it is rarely used for commercial production. Attempts to use Rhizopus sp. for lactic acid production through either hetero-fermentative or thermophilic conditions were not economical. Since almost 70% of the cost of its production is accounted by raw materials, R & D efforts are still focused to find economically attractive agri-products to serve as sources of carbon and complex nitrogen inputs to meet fastidious nutrient needs for microbial growth and lactic acid production. Therefore, need exists for using multi-pronged strategies for higher productivity. Its present production and consumption scenario is examined. Its optically active isomers and chemical structure permit its use for the production of several industrially important chemicals, health products (probiotics), food preservatives, and bio-plastics. In addition, its salts and esters appear to have a variety of applications.

The effect of cultivation conditions on the optical purity of L(+)-lactic acid produced by Rhizopus oryzae HZS6 was investigated. The isomeric composition of lactic acid was influenced by the supplementation of L(+)-lactic acid to fermentation medium. L(+)-isomer increased with the dosage, no D(-)-lactic acid was observed when the concentration of supplemented L(+)-lactic acid in matrix was > or = 1.5 g/L. However, the L(+)-lactic acid yield, biomass and glucose conversion rate decreased with the dosage. With the same method, the supplementation of L(+)-lactic to substrate had no influence on isomeric composition of lactic acid by Lactobacillus and Escherichia coli.

Acid-producing Rhizopus fungi from loog-pang, a traditional Thai fermented food, was screened to investigate its potential for use in industrial lactic acid production from starch. A thermotolerant strain, TISTR 3518, was isolated and characterized by its morphological, physiological, genetic and fermentation properties, and compared with its mesophilic isolates, TISTR 3514 and TISTR 3523. TISTR 3518 was characterized by shorter sporangiophores and smaller sporangia than the other isolates; however, apparent differences between the mesophilic isolates and the strain could not be clarified. Moreover, TISTR 3518 grew at 45 degrees C, whereas the others did not. The three isolates showed different profiles of oligosaccharide assimilation and organic acid production. Their rDNA ITS sequences indicated that TISTR 3518 is a strain of Rhizopus microsporus, and TISTR 3514 and TISTR 3523 are strains of Rhizopus oryzae. TISTR 3523 and TISTR 3518 mainly formed L-lactic acid from glucose, while TISTR 3514 primarily formed fumaric acid. Under thermotolerant conditions, R. microsporus TISTR 3518 showed higher glucoamylase activity than the others, suggesting this enzyme from TISTR 3518 is more thermostable than that from TISTR 3523. The strain formed higher amounts of L-lactic acid from starch at 40 degrees C compared to R. oryzae TISTR 3523. This is the first report on the production of optically active L-lactic acid from starch by a thermotolerant fungus and could potentially provide a good tool for transforming biomass resources to chemical materials.

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.

In order to carry out the process of the repeated intermittent L-lactic acid fermentation by self-immobilized Rhizopus oryzae, we investigated the effect of medium compositions on the morphology of Rhizopus oryzae. In submerged culture, fungi can be grown as broths of freely suspended mycelia and pellets or clumps, the pellets can be immobilized by themselves. The optimum medium composition for the first patch of fermentation was: 120 g/L glucose, 3 g/L NH4NO3, 0.14 g/L KH2PO4, 0.16 g/L NaH2PO4 (the concentration ratio of K+ and Na+ was 1:1). After 72 h fermentation, the physical form of Rhizopus oryzae was mostly uniform pellet with the diameter of 1.0-2.0 mm, the concentration of L-lactic acid was 100.8 g/L, and the conversion rate of glucose was 84%. During 16 batches of repeated fermentation, the L-lactic acid level was above 60.0 g/L and the conversion rate of glucose was 75%. The fermentation time of every batch was 24 h.

The fungus Rhizopus oryzae converts both glucose and xylose under aerobic conditions into chirally pure L+-lactic acid with by-products such as xylitol, glycerol, ethanol, carbon dioxide and fungal biomass. In this paper, we demonstrate that the production of lactic acid by R. oryzae CBS 112.07 only occurs under growing conditions. Deprivation of nutrients such as nitrogen, essential for fungal biomass formation, resulted in a cessation of lactic acid production. Complete xylose utilisation required a significantly lower C/N ratio (61/1) compared to glucose (201/1), caused by higher fungal biomass yields that were obtained with xylose as substrate. Decreasing the oxygen transfer rate resulted in decline of xylose consumption rates, whereas the conversion of glucose by R. oryzae was less affected. Both results were linked to the fact that R. oryzae CBS 112.07 utilises xylose via the two-step reduction/oxidation route. The consequences of these effects for R. oryzae as a potential lactic acid producer are discussed.

Spores of the filamentous fungus Rhizopus oryzae were entrapped in macroporous poly(vinyl alcohol) cryogel (PVA-cryogel). To prepare immobilised biocatalyst capable of producing L(+)-lactic acid (LA), the fungus cells were cultivated inside the carrier beads. The growth parameters and metabolic activity of the suspended (free) and immobilised cells producing LA in a batch process were comparatively investigated. The immobilised cells possessed increased resistance to high concentrations of accumulated product and gave much higher yields of LA in the iterative working cycle than the free cells did. Detailed kinetic analysis of the changes in the intracellular adenosine triphosphate concentration, specific rate of growth, substrate consumption and LA production showed that the fungus cells entrapped in PVA-cryogel are more attractive for biotechnological applications compared to the free cells.

In order to study the feasibility of commercial-scale L-lactic acid production by Rhizopus sp. MK-96-1196 using large scale airlift bioreactors (ALBs), a scale-up study from 0.003 m3 to 5 m3 ALB was carried out using oxygen transfer rate (OTR) as the scale-up criterion. Enhanced L-lactic acid production was achieved at OTRs higher than 0.28 (g-O2/l/h) irrespective of the scale of the bioreactor in question: in the range of 0.003 m3 to 5 m3, more than 90 g/lL-lactic acid was produced with a yield of approximately 80%, based on the initial glucose concentration. In future research, we plan to design an ALB greater than 3000 m3 (working volume: 2000 m3) for further studies on the production of L-lactic acid in large quantities.

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.

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 degrees 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.

Twenty-seven strains of Rhizopus oryzae accumulating predominantly lactic acid were shown to possess two ldh genes, ldhA and ldhB, encoding NAD-dependent lactate dehydrogenases. Variation in nucleotide sequence was identified for each gene from different strains, and similar phylogenetic trees were obtained based on the nucleotide sequences of both genes. The other 21 strains of R. oryzae accumulating predominantly fumaric and malic acids contained a single ORF of ldhB. Compared to the strains accumulating predominantly lactic acid, a lower degree of sequence divergence was found in ldhB, resulting in a separate cluster in the phylogenetic tree. The high similarity (>90%) spanning the ORF and adjacent regions demonstrates that ldhA and ldhB are derived from the same ancestor gene. The strains accumulating predominantly fumaric and malic acids lack functional ldhA, which plays a role in lactic acid synthesis and may form a lineage separated from the strains accumulating predominantly lactic acid in the genus Rhizopus.

Rhizopus arrhizus, strain DAR 36017, produced L(+)-lactic acid in a simultaneous saccharification and fermentation process using starch waste effluents. Lactic acid at 19.5-44.3 g l(-1) with a yield of 0.85-0.96 g g(-1) was produced in 40 h using 20-60 g starch l(-1). Supplementation of nitrogen source may be unnecessary if potato or corn starch waste effluent was used as a production medium.

A rotating fibrous-bed bioreactor (RFB) was developed for fermentation to produce L(+)-lactic acid from glucose and cornstarch by Rhizopus oryzae. Fungal mycelia were immobilized on cotton cloth in the RFB for a prolonged period to study the fermentation kinetics and process stability. The pH and dissolved oxygen concentration (DO) were found to have significant effects on lactic acid productivity and yield, with pH 6 and 90% DO being the optimal conditions. A high lactic acid yield of 90% (w/w) and productivity of 2.5 g/L.h (467 g/h.m(2)) was obtained from glucose in fed-batch fermentation. When cornstarch was used as the substrate, the lactic acid yield was close to 100% (w/w) and the productivity was 1.65 g/L.h (300 g/h.m(2)). The highest concentration of lactic acid achieved in these fed-batch fermentations was 127 g/L. The immobilized-cells fermentation in the RFB gave a virtually cell-free fermentation broth and provided many advantages over conventional fermentation processes, especially those with freely suspended fungal cells. Without immobilization with the cotton cloth, mycelia grew everywhere in the fermentor and caused serious problems in reactor control and operation and consequently the fermentation was poor in lactic acid production. Oxygen transfer in the RFB was also studied and the volumetric oxygen transfer coefficients under various aeration and agitation conditions were determined and then used to estimate the oxygen transfer rate and uptake rate during the fermentation. The results showed that the oxygen uptake rate increased with increasing DO, indicating that oxygen transfer was limited by the diffusion inside the mycelial layer., (Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 1-12, 2002.)

L-(+)-Lactic acid production was enhanced in a culture of Rhizopus oryzae by induction of a mycelial flocs morphology. By conventional culture the morphology of R. oryzae is that of a pellet-like cake; however, when mineral support and poly(ethylene oxide) are added to the culture, the morphology of R. oryzae takes on a cotton-like appearance. The formation of these cotton-like mycelial flocs was induced by the addition of 5 ppm poly(ethylene oxide) into a 12-14 h culture containing 3 g/L of the mineral support before the formation of the conventional pellet morphology. The cotton-like flocs were also formed in cultures grown in an air-lift bioreactor. This morphology allowed effective mass transfer inside the flocs and effective fluidity of culture broth in an air-lift bioreactor. L-(+)-Lactic acid concentration produced by mycelial flocs in an air-lift bioreactor, with the support and poly(ethylene oxide), was 104.6 g/L with a yield of 0.87 using 120 g/L of glucose as the substrate; for this culture without both, the concentration was 43.2 g/L. These results demonstrate that cotton-like mycelial flocs are the optimal morphology for use in the air-lift bioreactor culture of R. oryzae.