Pomegranate peel waste: a new substrate for citric acid production by Aspergillus niger in solid-state fermentation under non-aseptic conditions.

Citric acid production from dried and non-dried pomegranate peel wastes by the fungus Aspergillus niger B60 in solid-state fermentation (SSF) under non-aseptic conditions was investigated. The maximum amount of citric acid (278.5 g/kg dry peel) was achieved using dried (at 45 °C for 48 h) pulverized pomegranate peels with moisture content 75% and initial pH 8.0, after 8 days of fermentation at 25 °C. Under the same fermentation conditions, a higher amount of citric acid (306.8 g/kg dry peel) was observed during SSF of non-dried peels as a substrate. The addition of methanol as an inducer at a concentration of 3% (w/w) into the dried and non-dried pomegranate peel wastes increased the amount of citric acid to 300.7 and 351.5 g/kg dry peel, respectively. The non-dried pomegranate peel waste in SSF under non-aseptic conditions is a cheap and useful substrate for the commercial production of citric acid with low energy cost. The utilization of inexpensive agro-industrial wastes through SSF can contribute to achieve industrially feasible and environmentally sustainable bio-production of citric acid.

Carotene production from waste cooking oil by Blakeslea trispora in a bubble column reactor: The role of oxidative stress.

The oxidative stress induced by hydroperoxides and reactive oxygen species (ROS) during carotene production from waste cooking oil (WCO) and corn steep liquor (CSL) by the fungus Blakeslea trispora in a bubble column reactor was investigated. The specific activities of the intracellular enzymes superoxide dismutase (SOD) and catalase (CAT) as well as the micromorphology of the fungus were measured in order to study the response of the fungus to oxidative stress. The changes of the morphology of microorganism leaded to pellets formation and documented using a computerized image analysis system. As a consequence of the mild oxidative stress induced by hydroperoxides of WCO and ROS a significant increase in carotene production was obtained. The highest carotene concentration (980.0 mg/l or 51.5 mg/g dry biomass) was achieved in a medium consisted of CSL (80.0 g/L) and WCO (50.0 g/L) at an aeration rate of 5 vvm after 6 days of fermentation. In this case the carotenes produced consisted of ß-carotene (71%), γ-carotene (26%), and lycopene (3%). The strong oxidative stress in the fungus caused a significant increase of γ-carotene concentration. Bubble column reactor is a useful fermentation system for carotene production in industrial scale.

Waste cooking oil: A new substrate for carotene production by Blakeslea trispora in submerged fermentation.

The objective of this study was to evaluate a waste, waste cooking oil (WCO) as substrate for carotene production by Blakeslea trispora in shake flask culture. WCO was found to be a useful substrate for carotene production. B. trispora formed only pellets during fermentation. The oxidative stress in B. trispora induced by hydroperoxides and BHT as evidenced by increase of the specific activities of superoxide dismutase (SOD) and catalase (CAT) increased significantly the production of carotenes. The highest concentration of carotenes (2021 ± 75 mg/l or 49.3 ± 0.2 mg/g dry biomass) was obtained in culture grown in WCO (50.0 g/l) supplemented with CSL (80.0 g/l) and BHT (4.0 g/l). In this case the carotenes produced consisted of ß-carotene (74.2%), γ-carotene (23.2%), and lycopene (2.6%). The external addition in the above medium glucose, Span 80, yeast extract, casein acid hydrolysate, l-asparagine, thiamine. HCl, KH2PO4, and MgSO4·7H2O did not improve the production of carotenes.

The role of oxidative stress on carotene production by Blakeslea trispora in submerged fermentation.

In aerobic metabolism, reactive oxygen species (ROS) are formed during the fermentation that can cause oxidative stress in microorganisms. Microbial cells possess both enzymatic and non-enzymatic defensive systems that may protect cells from oxidative damage. The antioxidant enzymes superoxide dismutase and catalase are the two key defensive enzymes to oxidative stress. The factors that induce oxidative stress in microorganisms include butylated hydroxytoluene (BHT), hydrogen peroxide, metal ions, dissolved oxygen tension, elevated temperature, menadione, junglone, paraquat, liquid paraffin, introduction to bioreactors of shake flask inocula and synthetic medium sterilized at initial pH 11.0. Carotenes are highly unsaturated isoprene derivatives. They are used as antioxidants and as coloring agents for food products. In fungi, carotenes are derived via the mevalonate biosynthesis pathway. The key genes in carotene biosynthesis are hmgR, ipi, isoA, carG, carRA and carB. Among microorganisms, Βlakeslea trispora is the main microorganism used for the production of carotenes on the industrial scale. Currently, the synthetic medium is considered the superior substrate for the production of carotenes in a pilot plant scale. The fermentation systems used for the production of carotenes include shake flasks, stirred tank fermentor, bubble column reactor and flat panel photobioreactor. This review summarizes the oxidative stresses in microorganisms and it is focused on the current status of carotene production by B. trispora including oxidative stress induced by BHT, enhanced dissolved oxygen levels, iron ions, liquid paraffin and synthetic medium sterilized at an initial pH 11.0. The oxidative stress induced by the above factors increases significantly the production of carotenes. However, to further reduce the cost of carotene production, new biotechnological methods with higher productivity still need to be explored.

From cheese whey to carotenes by Blakeslea trispora in a bubble column reactor.

The effect of the aeration rate on carotene production from deproteinized hydrolyzed whey by Blakeslea trispora in a bubble column reactor was investigated. Aeration rate significantly affected carotene concentration and morphology of the fungus. Enhanced aeration caused change of the morphology of B. trispora from pellets with large projected area to pellets with small projected area. This morphological differentiation of the fungus was associated with a significant increase in carotene production. When deproteinized hydrolyzed whey was supplemented with 30 g/l Tween 80, 30 g/l Span 80, and 0.2 % (v/v) ß-ionone, the highest carotene productivity (55.5 mg/g dry biomass/day or 405.0 mg/l/day) was obtained at an aeration rate of 4 vvm. This is the highest carotene productivity that has been reported among the agro-industrial by-products up to date. In this case, the carotenes produced consisted of ß-carotene (67 %), γ-carotene (15 %), and lycopene (18 %).

Oxidative stress response of Blakeslea trispora induced by iron ions during carotene production in shake flask culture.

The adaptive response of the fungus Blakeslea trispora to the oxidative stress induced by iron ions during carotene production in shake flask culture was investigated. The culture response to oxidative stress was studied by measuring the specific activities of catalase (CAT) and superoxide dismutase (SOD). The addition of 1.0 mM of FeCl3 to the medium was associated with a mild oxidative stress as evidenced by remarkable increase of the specific activities of SOD and CAT. On the other hand, the addition 5.0 mM of FeCl3 caused a strong oxidative stress resulting in a drastic decrease in carotene concentration. The oxidative stress in B. trispora changed the composition of the carotenes and caused a significant increase of γ-carotene ratio. The highest concentration of carotenes (115.0 ± 3.5 mg/g dry biomass) was obtained in the basal medium without the addition of FeCl3 after 8 days of fermentation. In this case, the carotenes consisted of ß-carotene (46.3 %), γ-carotene (40.1 %), and lycopene (13.6 %). The addition of 1.0 mM of FeCl3 into the medium did not change the concentration of carotenes. But, the composition of carotenes was changed with a drastic increase of γ-carotene ratio (61.6 %) and a decrease in ß-carotene and lycopene ratio (31.2 and 7.2 %, respectively).

Stimulation of the biosynthesis of carotenes by oxidative stress in Blakeslea trispora induced by elevated dissolved oxygen levels in the culture medium.

The adaptive response of the fungus Blakeslea trispora to the oxidative stress induced by elevated dissolved oxygen concentrations during carotene production was investigated by measuring the specific activities of catalase (CAT) and superoxide dismutase (SOD) and the micromorphology of the fungus using a computerized image analysis system. Changes in the ratio of the volume of air (V(a)) over the medium and the volume of medium (V(m)) in the flask caused changes of the morphology of microorganism from clumps to pellets and increases in the specific activities of CAT and SOD. The oxidative stress in B. trispora resulted in a significant increase in carotene production, and a maximum proportion of ß-carotene (60%), γ-carotene (50%), and lycopene (10%) (as percentages of total carotenes) was observed at a ratio V(a)/V(m) of 15.7, 4.0 and 1.5, respectively. The highest concentration of carotenes (115.0mg/g dry biomass) was obtained in V(a)/V(m) ratio of 9.0.

Autolysis of Blakeslea trispora during carotene production from cheese whey in an airlift reactor.

The phenomenon of autolysis in Blakeslea trispora during carotene production from deproteinized hydrolyzed whey in an airlift reactor was investigated. The process of cellular autolysis was studied by measuring the changes in carotene concentration, dry biomass, residual sugars, pH, intracellular protein, specific activity of the hydrolytic enzymes (proteases, chitinase), and micromorphology of the fungus using a computerized image analysis system. All these parameters were useful indicators of autolysis, but image analysis was found to be the most useful indicator of the onset and progress of autolysis in the culture. Autolysis of B. trispora began early in the growth phase, continued during the stationary phase, and increased significantly in the decline phase. The morphological differentiation of the fungus was a result of the degradation of the cell membrane by hydrolytic enzymes. The biosynthesis of carotenes was carried out in the exponential phase, where the phenomenon of autolysis was not intense.

Oxidative stress response and morphological changes of Blakeslea trispora induced by butylated hydroxytoluene during carotene production.

The adaptive response of the fungus Blakeslea trispora to the oxidative stress induced by butylated hydroxytoluene (BHT) during carotene production in shake flask culture was investigated. The culture response to oxidative stress was studied by measuring the specific activities of catalase (CAT) and superoxide dismutase (SOD) and the micromorphology of the fungus using a computerized image analysis system. The addition of exogenous BHT to the medium caused changes of the morphology of microorganism from aggregates with large projected area to aggregates with small projected area. This morphological differentiation of the fungus was associated with high oxidative stress as evidenced by remarkable increase of the specific activities of CAT and SOD. The oxidative stress in B. trispora resulted in a fivefold increase of carotene production. The highest concentration of carotenes (125.0 mg/g dry biomass) was obtained in culture grown in medium supplemented with 20 mM of BHT.

Production of beta-carotene from beet molasses by Blakeslea trispora in stirred-tank and bubble column reactors: development of a mathematical modeling.

The effect of aeration rate and agitation speed on beta-carotene production from molasses by Blakeslea trispora in a stirred-tank fermentor and optimization of the production of the pigment in a bubble column reactor were investigated. In addition, a central composite design was employed to determine the maximum beta-carotene concentration at optimum values for the process variables (aeration rate, sugar concentration, linoleic acid, kerosene). By image analysis of the morphology of the fungus, a quantitative characterization of the hyphae and zygospores formed was obtained. The hyphae were differentiated to intact hyphae, vacuolated hyphae, evacuated cells and degenerated hyphae. An increased proportion of zygospores was correlated to high beta-carotene production. In the stirred-tank fermentor, the highest concentration of the carotenoid pigment (92.0 mg/L) was obtained at an aeration rate of 1.5 vvm and agitation speed of 60 rpm. In the bubble column reactor, the aeration rate and concentration of sugars, linoleic acid, kerosene, and antioxidant significantly affected the production of beta-carotene. In all cases, the fit of the model was found to be good. Aeration rate, sugar concentration, linoleic acid, and kerosene had a strong positive linear effect on beta-carotene concentration. Moreover, the concentration of the pigment was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions. Maximum beta-carotene concentration (360.2 mg/L) was obtained in culture grown in molasses solution containing 5% (w/v) sugar supplemented with linoleic acid (37.59 g/L), kerosene (39.11 g/L), and antioxidant (1.0 g/L).

Production and characterization of pullulan from beet molasses using a nonpigmented strain of Aureobasidium pullulans in batch culture.

The production of pullulan from beet molasses by a pigment-free strain of Aztreobasidium pullulans on shake-flask culture was investigated. Combined pretreatment of molasses with sulfuric acid and activated carbon to remove potential fermentation inhibitors present in molasses resulted in a maximum pullulan concentration of 24 g/L, a biomass dry wt of 14 g/L, a pullulan yield of 52.5%, and a sugar utilization of 92% with optimum fermentation conditions (initial sugar concentration of 50 g/L and initial pH of 7.0). The addition of other nutrients as carbon and nitrogen supplements (olive oil, ammonium sulfate, yeast extract) did not further improve the production of the exopolysaccharides. Structural characterization of the isolated polysaccharides from the fermentation broths by 13C-nuclear magnetic resonance spectroscopy and pullulanase digestion combined with size-exclusion chromatography confirmed the identity of pullulan and the homogeneity (>93% dry basis) of the elaborated polysaccharides by the microorganism. Using multiangle laser light scattering and refractive index detectors in conjunction with high-performance size-exclusion chromatography molecular size distributions and estimates of the molecular weight (Mw = 2.1-4.1 x 10(5)), root mean square of the radius of gyration (R = 30-38 nm), and polydispersity index (Mw/Mn = 1.4-2.4) were obtained. The fermentation products of molasses pretreated with sulfuric acid and/or activated carbon were more homogeneous and free of contaminating proteins. In the concentration range of 2.8-10.0 (w/v), the solution's rheologic behavior of the isolated pullulans was almost Newtonian (within 1 and 1200 s(-1) at 20 degrees C); a slight shear thinning was observed at 10.0 (w/v) for the high molecular weight samples. Overall, beet molasses pretreated with sulfuric acid and activated carbon appears as an attractive fermentation medium for the production of pullulan by A. pullulans.