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.     

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.     

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.     

Effects of Cultivation Parameters on the Morphology of Rhizopus arrhizus and the Lactic Acid Production in a Bubble Column Reactor

The use of filamentous Rhizopus for lactic acid production is facing a challenge due to its low yield mainly caused by the difficulty to control its morphology in submerged fermentation processes. This study was aimed at investigating the impacts of cultivation parameters on the morphology of Rhizopus arrhizus DAR 36017 and lactic acid production using waste potato starch in a laboratory scale bubble column reactor (BCR). The fungal morphology was significantly influenced by carbon sources, process pH, starch concentrations, sparger designs and aeration rates. The favorable morphology for lactic acid production was a freely dispersed small pellet, which was achieved under operation conditions at pH 5.0–6.0, starch concentrations of 60–120 g/L and aeration rates of 0.2–0.8 vvm using a sintered stainless steel disc sparger. Optimal cultivation conditions at pH 6.0 and an aeration rate of 0.4 vvm resulted in the formation of freely dispersed small pellets and 103.8 g/L lactic acid with a yield of 87 % from 120 g/L liquefied potato starch in 48 h. The overall results in terms of lactic acid yield and productivity are comparable to those reported in previous studies using immobilized Rhizopus cells in batch fermentations.     

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.     

Production of lactic acid and byproducts from waste potato starch by <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis>: role of nitrogen sources

Summary Lactic acid was produced by Rhizopus arrhizus using waste potato starch as the substrate. The aim of this study was to identify the role of nitrogen sources and their impact on the formation of lactic acid and associated byproducts. Ammonium sulphate, ammonium nitrate, urea, yeast extract and peptone were assessed in conjunction with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio favoured the production of fumaric acid. Ammonium nitrate appeared to be the most suitable nitrogen source for achieving a high and stable lactic acid yield, and minimizing the production of byproducts such as biomass and ethanol, while urea proved to be the least favourable nitrogen source. Yeast extract and peptone appeared to improve fungal cell growth. The kinetics data revealed that a high concentration of ammonium nitrate enhanced the lactic acid productivity. The maximum lactic acid concentration of 36.4 g/l, representing a yield of 91%, was obtained with addition of 0.909 g/l ammonium nitrate in 32 h.     

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.     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Bioconversion of cheese whey permeate into fungal oil by <Emphasis Type="Italic">Mucor circinelloides</Emphasis>

Background

Oleaginous fungi are efficient tools to convert agricultural waste streams into valuable components. The filamentous fungus Mucor circinelloides was cultivated in whey permeate, a byproduct from cheese production, to produce an oil-rich fungal biomass. Response surface methodology was used to optimize the fermentation conditions such as pH and temperature for increased biomass yield and lipid accumulation. Quantification and characterization of the fungal biomass oil was conducted.

Results

Upstream lactose hydrolysis of the whey permeate increased the biomass yield from 2.4 to 7.8 (g dry biomass/L) compared to that of non-hydrolyzed whey permeate. The combination of low pH (4.5) and pasteurization minimized microbial competition, thus favoring fungal growth. A central composite rotatable design was used to evaluate the effects of temperature (22.4–33.6 °C) and a lower pH range (3.6–4.7) on biomass yield and composition. The highest biomass yield and oil content was observed at high temperature (33.6 °C), while the pH range evaluated had a less pronounced effect. The predictive model was validated at the optimal conditions of 33.6 °C and pH 4.5. The fungal biomass yield plateaued at 9 g dry cell weight per liter, while the oil content and lipid yield reached a maximum of 24% dry biomass and 2.20 g/L, respectively, at 168 h. Triacylglycerides were the major lipid class (92%), which contained predominantly oleic (41%), palmitic (23%), linoleic (11%), and γ-linolenic acid (9%).

Conclusions

This study provided an alternative way of valorization of cheese whey permeate by using it as a substrate for the production of value-added compounds by fungal fermentation. The fatty acid profile indicates the suitability of M. circinelloides oil as a potential feedstock for biofuel production and nutraceutical applications.

     

Enhancement and modeling of microparticle-added <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactic acid production

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.     

Lactic acid production from xylose by the fungus Rhizopus oryzae

Lignocellulosic biomass is considered nowadays to be an economically attractive carbohydrate feedstock for large-scale fermentation of bulk chemicals such as lactic acid. The filamentous fungus Rhizopus oryzae is able to grow in mineral medium with glucose as sole carbon source and to produce optically pure l(+)-lactic acid. Less is known about the conversion by R. oryzae of pentose sugars such as xylose, which is abundantly present in lignocellulosic hydrolysates. This paper describes the conversion of xylose in synthetic media into lactic acid by ten R. oryzae strains resulting in yields between 0.41 and 0.71 g g⁻¹. By-products were fungal biomass, xylitol, glycerol, ethanol and carbon dioxide. The growth of R. oryzae CBS 112.07 in media with initial xylose concentrations above 40 g l⁻¹ showed inhibition of substrate consumption and lactic acid production rates. In case of mixed substrates, diauxic growth was observed where consumption of glucose and xylose occurred subsequently. Sugar consumption rate and lactic acid production rate were significantly higher during glucose consumption phase compared to xylose consumption phase. Available xylose (10.3 g l⁻¹) and glucose (19.2 g l⁻¹) present in a mild-temperature alkaline treated wheat straw hydrolysate was converted subsequently by R. oryzae with rates of 2.2 g glucose l⁻¹ h⁻¹ and 0.5 g xylose l⁻¹ h⁻¹. This resulted mainly into the product lactic acid (6.8 g l⁻¹) and ethanol (5.7 g l⁻¹).     

Improvement of Omega-3 Docosahexaenoic Acid Production by Marine Dinoflagellate Crypthecodinium cohnii Using Rapeseed Meal Hydrolysate and Waste Molasses as Feedstock

Rapeseed meal and waste molasses are two important agro-industrial by-products which are produced in large quantities. In this study, solid state fermentation and fungal autolysis were performed to produce rapeseed meal hydrolysate (RMH) using fungal strains of Aspergillus oryzae, Penicillium oxalicum and Neurospora crassa. The hydrolysate was used as fermentation feedstock for heterotrophic growth of microalga Crypthecodinium cohnii that produce docosahexaenoic acid (DHA). The addition of waste molasses as a supplementary carbon source greatly increased the biomass and DHA yield. In the batch fermentations using media composed of diluted RMH (7%) and 1-9% waste molasses, the highest biomass concentration and DHA yield reached 3.43 g/L and 8.72 mg/L, respectively. The algal biomass produced from RMH and molasses medium also had a high percentage of DHA (22-34%) in total fatty acids similar to that of commercial algal biomass. RMH was shown to be rich in nitrogen supply comparable to the commercial nitrogen feedstock like yeast extract. Using RMH as sole nitrogen source, waste molasses excelled other carbon sources and produced the highest concentration of biomass. This study suggests that DHA production of the marine dinoflagellate C. cohnii could be greatly improved by concomitantly using the cheap by-products rapeseed meal hydrolysate and molasses as alternative feedstock.     

<Emphasis Type="Italic">Rhizopus arrhizus</Emphasis> – a producer for simultaneous saccharification and fermentation of starch waste materials to l(+)-lactic acid

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.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

d‐lactic acid production from renewable lignocellulosic biomass via genetically modified Lactobacillus plantarum

d ‐lactic acid is of great interest because of increasing demand for biobased poly‐lactic acid (PLA). Blending poly‐l ‐lactic acid with poly‐d ‐lactic acid greatly improves PLA's mechanical and physical properties. Corn stover and sorghum stalks treated with 1% sodium hydroxide were investigated as possible substrates for d ‐lactic acid production by both sequential saccharification and fermentation and simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Cellic CTec2) was used for hydrolysis of lignocellulosic biomass and an l ‐lactate‐deficient mutant strain Lactobacillus plantarum NCIMB 8826 ldhL1 and its derivative harboring a xylose assimilation plasmid (ΔldhL1‐pCU‐PxylAB) were used for fermentation. The SSCF process demonstrated the advantage of avoiding feedback inhibition of released sugars from lignocellulosic biomass, thus significantly improving d ‐lactic acid yield and productivity. d ‐lactic acid (27.3 g L^?1) and productivity (0.75 g L^?1 h^?1) was obtained from corn stover and d ‐lactic acid (22.0 g L^?1) and productivity (0.65 g L^?1 h^?1) was obtained from sorghum stalks using ΔldhL1‐pCU‐PxylAB via the SSCF process. The recombinant strain produced a higher concentration of d ‐lactic acid than the mutant strain by using the xylose present in lignocellulosic biomass. Our findings demonstrate the potential of using renewable lignocellulosic biomass as an alternative to conventional feedstocks with metabolically engineered lactic acid bacteria to produce d ‐lactic acid. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:271–278, 2016     

少根根霉产孢能力、变种与发酵产物的相关性

[目的]探究少根根霉不同生态环境、不同地理位置、不同产孢能力和不同分类学变种的发酵产物多样性及相关性,为工业生产提供指导.[方法]代表性的68株少根根霉菌株于糯米培养基中进行液态发酵,利用高效液相色谱法测定各种发酵产物浓度,计算发酵产物间的Pearson相关系数,通过多因素方差分析和主成分分析解析各类菌株与发酵特性的相...     

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.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using semicontinuous fermentation in bioreactor

Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO₃ addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation with 4% spore suspension, CaCO₃ added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH₄)₂SO₄, 2; KH₂PO₄, 0.1; ZnSO₄·7H₂O, 0.33; MgSO₄·7H₂O, 0.15; CaCO₃, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases (LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor, semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle; in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h).     

Production of lactic acid from hemicellulose extracts by <Emphasis Type="Italic">Bacillus coagulans</Emphasis> MXL-9

Bacillus coagulans MXL-9 was found capable of growing on pre-pulping hemicellulose extracts, utilizing all of the principle monosugars found in woody biomass. This organism is a moderate thermophile isolated from compost for its pentose-utilizing capabilities. It was found to have high tolerance for inhibitors such as acetic acid and sodium, which are present in pre-pulping hemicellulose extracts. Fermentation of 20 g/l xylose in the presence of 30 g/l acetic acid required a longer lag phase but overall lactic acid yield was not diminished. Similarly, fermentation of xylose in the presence of 20 g/l sodium increased the lag time but did not affect overall product yield, though 30 g/l sodium proved completely inhibitory. Fermentation of hot water-extracted Siberian larch containing 45 g/l total monosaccharides, mainly galactose and arabinose, produced 33 g/l lactic acid in 60 h and completely consumed all sugars. Small amounts of co-products were formed, including acetic acid, formic acid, and ethanol. Hemicellulose extract formed during autohydrolysis of mixed hardwoods contained mainly xylose and was converted into lactic acid with a 94% yield. Green liquor-extracted hardwood hemicellulose containing 10 g/l acetic acid and 6 g/l sodium was also completely converted into lactic acid at a 72% yield. The Bacillus coagulans MXL-9 strain was found to be well suited to production of lactic acid from lignocellulosic biomass due to its compatibility with conditions favorable to industrial enzymes and its ability to withstand inhibitors while rapidly consuming all pentose and hexose sugars of interest at high product yields.     

Role of the pectinolytic enzyme in the lactic acid fermentation of potato pulp by<Emphasis Type="Italic"> Rhizopus oryzae</Emphasis>

Rhizopus oryzae strain NBRC 4707 produced lactic acid and ethanol more efficiently than strain NRRL 395 in potato pulp, an agricultural by-product of the starch industry. The two strains developed comparable activities of xylanase, cellulase, -amylase, and glucoamylase, while the polygalacturonase activity of strain NBRC 4707 was double that of strain NRRL 395. The addition of commercial pectinase enhanced the formation of metabolites, suggesting that the degradation of pectic substances determines the fermentation of potato pulp by R. oryzae. Orange and apple peel were more effective in the induction of polygalacturonase activity than potato pulp, sugarbeet pulp, or wheat bran when used as a principal carbon source for fungal growth in a solid-state culture. The fungal cells in both types of fruit peel stimulated the fermentation of potato pulp and increased the quantity of lactic acid and ethanol to higher levels than those in other agricultural by-products.