Itaconic acid production by immobilizedAspergillus terreus from xylose and glucose

Summary Aspergillus terreus NRRC 1960 spores were entrapped in calcium alginate gel beads or alternotely the fungal mycelium was immobilized either on Celite R-626 or in agar gel cubes, and the biocatalyst was employed both in repeated batch and in continuous column reactors to produce itaconic acid from D-xylose or D-glucose. The highest itaconic acid yield obtained in a submerged culture batch fermentation was 54.5% based on total initial glucose (55 g/l) with a volumetric productivity of 0.32 g/l h, and 44.8% from xylose (67 g/l) with a productivity of 0.20 g/l h. In a repeated batch fermentation mycelium immobilized in agar gel had a productivity of 0.112 g/l h, and mycelium grown from spores immobilized in calcium alginate gel 0.06 g/l h, both from xylose (60 g/l). With the best immobilized biocatalyst system used employing Celite R-626 as a carrier, volumetric productivities of 1.2 g/l h from glucose and 0.56 g/l h from xylose (both at 60 g/l) were obtained in continuous column operation for more than 2 weeks.     

High-yield production of citric acid by Yarrowia lipolytica on glycerol in repeated-batch bioreactors

An acetate negative mutant of Yarrowia lipolytica Wratislavia AWG7 was found to be suitable for the production of high amounts of citric acid in long-term repeated-batch cultures. When 40% of fresh replaced medium was fed, this strain produced 154 g l⁻¹, on average, which corresponded to a 0.78 g g⁻¹ yield and a productivity of 1.05 g l⁻¹ h⁻¹. The activity of the culture remained stable for more than 1,650 h, i.e., 16 cycles of the repeated-batch bioreactors.     

Itaconic acid production in microorganisms

Itaconic acid, 2-methylidenebutanedioic acid, is a precursor of polymers, chemicals, and fuels. Many fungi can synthesize itaconic acid; Aspergillus terreus and Ustilago maydis produce up to 85 and 53 g l^?1, respectively. Other organisms, including Aspergillus niger and yeasts, have been engineered to produce itaconic acid. However, the titer of itaconic acid is low compared with the analogous major fermentation product, citric acid, for which the yield is > 200 g l^?1. Here, we review two types of pathway for itaconic acid biosynthesis as well as recent advances by metabolic engineering strategies and process optimization to enhance itaconic acid productivity in native producers and heterologous hosts. We also propose further improvements to overcome existing problems.     

Fermentative hydrogen production from fresh leachate in batch and continuous bioreactors

This research for the first time investigated hydrogen production from the fresh leachate originated from municipal solid wastes. We found that fermentation of the leachate generated H₂ and was very much enhanced in the presence of extra phosphate in the batch reactor. The continuous expanded granular sludge bed (EGSB) reactor started to generate H₂ at day 20 and continued to 176 days with 120 mg/l of extra phosphate present. The highest chemical oxygen demand (COD) removal efficiency (66.9%) was achieved at liquid up-flow velocity of 3.7 m/h and hydraulic retention time of 12 h. Under proposed optimal operation conditions, the mean H₂ production rate reached up to 2155 ml/(l day). We also found that over 80% liquid metabolites were acetic acid and ethanol, suggesting the ethanol-type fermentation was dominant in the bioreactor. These findings indicate that the fresh leachate can be used as the source for continuous hydrogen production.     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

Studies on citric acid production with immobilized Yarrowia lipolytica in repeated batch and continuous air-lift bioreactors

Citric acid was produced from glucose in repeated-batch shake-flask and continuous air-lift cultivations by calcium-alginate-immobilized Yarrowia lipolytica A-101 yeast. The medium composition was systematically studied in a batch system by using experimental design and empiric modelling. The highest citric acid product concentration of 39 g/l was reached with a medium containing 150 g/l of glucose, 0.105 g/l of potassium dihydrogen phosphate, 0.84 g/l of magnesium sulphate and 21 mg/l of copper sulphate (5.2 mg/l of copper). The results were further improved by hardening the alginate carrier beads with glutaraldehyde, and by activation of the immobilized biocatalyst in a nutrient solution. In continuous air-lift bioreactors with varying height-to-diameter ratio the highest productivity of 350 mg/l per hour with a dilution rate of 0.023 l/h and a citric acid product concentration of 12 g/l was reached with a ratio of 3. Correspondence to: H. Kautola     

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).     

Efficient production of L-lactic acid from xylose by Pichia stipitis

Microbial conversion of renewable raw materials to useful products is an important objective in industrial biotechnology. Pichia stipitis, a yeast that naturally ferments xylose, was genetically engineered for l-(+)-lactate production. We constructed a P. stipitis strain that expressed the l-lactate dehydrogenase (LDH) from Lactobacillus helveticus under the control of the P. stipitis fermentative ADH1 promoter. Xylose, glucose, or a mixture of the two sugars was used as the carbon source for lactate production. The constructed P. stipitis strain produced a higher level of lactate and a higher yield on xylose than on glucose. Lactate accumulated as the main product in xylose-containing medium, with 58 g/liter lactate produced from 100 g/liter xylose. Relatively efficient lactate production also occurred on glucose medium, with 41 g/liter lactate produced from 94 g/liter glucose. In the presence of both sugars, xylose and glucose were consumed simultaneously and converted predominantly to lactate. Lactate was produced at the expense of ethanol, whose production decreased to approximately 15 to 30% of the wild-type level on xylose-containing medium and to 70 to 80% of the wild-type level on glucose-containing medium. Thus, LDH competed efficiently with the ethanol pathway for pyruvate, even though the pathway from pyruvate to ethanol was intact. Our results show, for the first time, that lactate production from xylose by a yeast species is feasible and efficient. This is encouraging for further development of yeast-based bioprocesses to produce lactate from lignocellulosic raw material.     

Itaconic acid production by immobilizedAspergillus terreus on sucrose medium

Summary The itaconic acid production by immobilizedAspergillus terreus TTK 200-5-3 mycelium was optimized in shake flask fermentations using statistical experimental design and empirical modelling. The maximum itaconic acid concentration was calculated to be 13.3 g/l in the investigated experimental area when initial sucrose concentration was 10%, ammonium nitrate concentration 0.275% and initial pH 3. The itaconic acid product concentration using immobilized mycelium was about double of that obtained with the free mycelium.     

Itaconic acid production by Aspergillus terreus on raw starchy materials

Starchy materials such as corn starch, soft wheat flour, potato flour, cassava flour, sorghum starch, sweet potato and industrial potato flours, either acid or enzymatically hydrolysed, were used as substrates for itaconic acid production by Aspergillus terreus NRRL 1960. Both production and yield were highest on corn starch (18·4 g l⁻¹ and 34·0%, respectively). The degree of hydrolysis had a great influence on acid production which was highest when corn starch was saccharified at 85 DE (dextrose equivalent). In a 3 litre benchtop fermenter, itaconic acid production and productivity were 19·8 g l⁻¹ and 0·13 g l⁻¹ h⁻¹, respectively.     

Fumaric acid production from xylose by immobilized Rhizopus arrhizus cells

Summary The production of fumaric acid by immobilized Rhizopus arrhizus TKK 204-1-1a mycelium was optimized in batch fermentations using statistical experimental design and empiric modelling. The maximum fumaric acid concentration was obtained at a xylose concentration of about 6% and a carbon:nitrogen ratio of about 160. In repeated batch fermentations with immobilized cells the highest volumetric productivity of fumaric acid reached was 87 mg/l per hour when the initial xylose concentration was 10%, the C:N ratio 160 and the residence time 1.75 days. The maximum product concentration was 16.4 g/l when the initial xylose concentration was 10%, the C:N ratio 160 and the residence time 10.25 days. The maximum yield from initial xylose (6.47%) was 23.7% with a product concentration of 15.3 g/l and volumetric productivity of 71 mg/l per hour at a residence time of 9 days and a C:N ratio of 188.3. Immobilization could increase the fumaric acid concentration to a level 3.4 times higher than that produced by free cells.     

Enhanced production of xylose from corncob hydrolysis with oxalic acid as catalyst

Bioprocess and Biosystems Engineering - The acid-catalyzed treatment was a conventional process for xylose production from corncob. To increase the release of xylose and to reduce the by-products...     

Two stirred-tank bioreactors in series enable continuous production of alcohols from carbon monoxide with Clostridium carboxidivorans

Bioprocess and Biosystems Engineering - Microbial batch production of alcohols by fermentation of CO-rich gases with Clostridia is limited by low volumetric productivities due to the need for...     

Itaconic acid production by immobilized Aspergillus terreus with varied metal additions

Summary The effect of trace and alkaline metals on itaconic acid production by polyurethane-foam-immobilized Aspergillus terreus was examined in repeated shake-flask cultures according to a statistical experimental design. An increase in the glucose or copper concentration increased the need for earth alkaline metals. The experimentally obtained highest itaconic acid concentration of 51 g/l from 15% glucose with a total productivity of 3.67 g/l per day was reached during the first 14-day batch fermentation. In the fourth batch the calculated highest itaconic acid concentration of 19 g/l was reached with 25% glucose, 5 g/l of magnesium sulphate, 13 mg/l of copper sulphate and 10 g/l of calcium chloride. The immobilization of the mycelium increased the itaconic acid concentration obtained by as much as eightfold.Offprint requests to: H. Kautola     

The performance of serial bioreactors in rapid continuous production of ethanol from dilute-acid hydrolyzates using immobilized cells

The performance of single, and series of, continuous stirred-tank (CSTBR) and fluidized-bed bioreactor (FBBR) in anaerobic continuous cultivation of glucose in defined media and dilute-acid hydrolyzates at dilution rates 0.22, 0.43, 0.65 and 0.86 h(-1) using immobilized Saccharomyces cerevisiae CBS 8066, was investigated. While the single CSTBR and FBBR could not take up more than 77% and 92% of glucose in a defined medium at dilution rate 0.86 h(-1), addition of the second bioreactor decreased the residual glucose to less than 1.1% of the incoming sugar. A similar trend was obtained in cultivation of dilute-acid hydrolyzates. A CSTBR could take up 75% and 54% of the initial fermentable sugars at dilution rates 0.43 and 0.86 h(-1), while the addition of the FBBR improved the assimilation of the sugars to 100% and 86%, respectively. The ethanol yields from the hydrolyzate were between 0.41 and 0.48 g/g in all the experiments. The specific and volumetric ethanol productivities were 1.13 g/gh and 5.98 g/Lh for the single bioreactor and 0.98 g/gh and 5.49 g/Lh for the serial bioreactor at the highest dilution rate, respectively. Glycerol was the only important by-product in terms of concentration, and yielded 0.05-0.07 g/g from the hydrolyzate. From the initial 3.98 g/L acetic acid present in the hydrolyzate, 0.1-0.8 g/L was assimilated by the cells. The yeast cells were accumulated close to the surface of the beads. While the cells had a dry-weight concentration of 129 g/L close to the surface of the beads, the concentration in the core was only 13 g/L.     

Development of semi-continuous and continuous flow bioreactors for the high level production of chloroperoxidase

Summary Continuous and semi-continuous flow bioreactors have been developed for the production of chloroperoxidase, which is secreted by the fungusCaldariomyces fumago. The semi-continuous system facilitated the harvest of mycelium-free supernatant for 200 days from a single inoculum, with enzyme levels greater than 600 mg/l during the first 80 days. The continuous flow bioreactor yielded essentially pure enzyme at the rate of 117 mg/day.     

Electricity production from xylose in fed-batch and continuous-flow microbial fuel cells

A medium-scale (0.77 l) air-cathode, brush-anode microbial fuel cell (MFC) operated in fed-batch mode using xylose (20 mM) generated a maximum power density of 13 +/- 1 W/m(3) (673 +/- 43 mW/m(2)). Xylose was rapidly removed (83.5%) within 8 h of a 60-h cycle, with 42.1% of electrons in intermediates (8.5 +/- 0.2 mM acetate, 5.9 +/- 0.01 mM ethanol, 4.3 +/- 0.1 mM formate, and 1.3 +/- 0.03 mM propionate), 9.1% captured as electricity, 16.1% in the remaining xylose, and 32.7% lost to cell storage, biomass, and other processes. The final Coulombic efficiency was 50%. At a higher initial xylose concentration (54 mM), xylose was again rapidly removed (86.9% within 24 h of a 116-h cycle), intermediates increased in concentration (18.4 +/- 0.4 mM acetate, 7.8 +/- 0.4 mM ethanol and 2.1 +/- 0.2 mM propionate), but power was lower (5.2 +/- 0.4 W/m(3)). Power was increased by operating the reactor in continuous flow mode at a hydraulic retention time of 20 h (20 +/- 1 W/m(3)), with 66 +/- 1% chemical oxygen demand removal. These results demonstrate that electricity generation is sustained over a cycle primarily by stored substrate and intermediates formed by fermentation and that the intermediates produced vary with xylose loading.     

L-lactate production from xylose employingLactococcus lactis IO-1

Summary The growth, substrate utilisation and L-lactate production ofLactococcus lactis IO-1 were examined on xylose, and glucose and xylose media. The yield of lactate on xylose was 0.47 g lactate/g xylose at an initial xylose concentration of 51.2 g/l and the _max was 0.72 h^–1. Xylose cultures were more susceptible to lactate inhibition than were glucose cultures but showed similar kinetic behaviour. The organism was capable of complete sugar utilisation when grown on a mixture of 20 g/l xylose and 20 g/l glucose and synthesised 0.66 g lactate/g sugar.