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The goals of this study were to optimize the medium formulation for enhanced production of Aspergillus niger inulinase using Plackett–Burman Design (PBD) and to model the fermentation in optimal medium formulation. Results indicated that (NH4)2SO4 (negative effect), yeast extract and peptone (positive effect) were determined as significant factors affecting the inulinase production. Different media including Medium A (non-enriched), Medium B (contains both negative and positive factors) and Medium C (contains only positive factors) were formed and inulinase fermentations were performed. Findings showed that the best nutritional formulation was Medium C, which yielded to be 1011.02 U/mL, 834.28 U/mL, 1.22, 4383.44 U/mg, 4186 U/mg, 158.49 U/mL/day, 128.60 U/mL/day and 94.54% of PInulinase, SInulinase, I/S ratio, SInulinase, SSucrase, QInulinase, QSucrase and SUY, respectively. Additionally, fungal growth, enzyme or protein production and substrate consumption were modeled using the logistic model, Luedeking–Piret model, and modified Luedeking–Piret model, respectively, and found that enzyme or protein production was non-growth associated. Besides, maintenance value (Z) was lower than γ value, indicating that A. niger mainly utilizes the sugars for enzyme production and fungal growth. Consequently, optimum medium composition was successfully determined by PBD and also the kinetic models fitted the experimental data very well with high regression coefficient.

Inulinases are used for the production of high-fructose syrup and fructooligosaccharides, and are widely utilized in food and pharmaceutical industries. In this study, different carbon sources were screened for inulinase production by Aspergillus niger in shake flask fermentation. Optimum working conditions of the enzyme were determined. Additionally, some properties of produced enzyme were determined [activation (Ea)/inactivation (Eia) energies, Q10 value, inactivation rate constant (kd), half-life (t1/2), D value, Z value, enthalpy (ΔH), free energy (ΔG), and entropy (ΔS)]. Results showed that sugar beet molasses (SBM) was the best in the production of inulinase, which gave 383.73 U/mL activity at 30 °C, 200 rpm and initial pH 5.0 for 10 days with 2% (v/v) of the prepared spore solution. Optimum working conditions were 4.8 pH, 60 °C, and 10 min, which yielded 604.23 U/mL, 1.09 inulinase/sucrase ratio, and 2924.39 U/mg. Additionally, Ea and Eia of inulinase reaction were 37.30 and 112.86 kJ/mol, respectively. Beyond 60 °C, Q10 values of inulinase dropped below one. At 70 and 80 °C, t1/2 of inulinase was 33.6 and 7.2 min; therefore, inulinase is unstable at high temperatures, respectively. Additionally, t1/2, D, ΔH, ΔG values of inulinase decreased with the increase in temperature. Z values of inulinase were 7.21 °C. Negative values of ΔS showed that enzymes underwent a significant process of aggregation during denaturation. Consequently, SBM is a promising carbon source for inulinase production by A. niger. Also, this is the first report on the determination of some properties of A. niger A42 (ATCC 204,447) inulinase.

Air flow rate and agitation speed for inulinase production by Kluyveromyces marxianus were optimized based on metabolic heat release profiles. Shear stress and oxygen transfer (kLa) values were compared to assess the effects of aeration and agitation. At agitation rates of ≤ 100 rpm, the oxygen mass transfer rates were small and eventually led to less inulinase production, but at agitation rates > 150 rpm, loss of biomass resulted in less inulinase activity. Bio-reaction calorimeter (BioRc1e) experiment with aeration rates ≤ 0.5 lpm showed low kLa while at 1.5 lpm frothing of reactor contents caused loss of biomass and inulinase activity. The optimum conditions for aeration and agitation rate for K. marxianus in BioRc1e were 1 lpm and 150 rpm. Heat yield values obtained for the substrate, product and biomass reinstated the ongoing metabolic process. The heat release pattern could be a promising tool for optimization of bioprocess and in situ monitoring, with a possibility of interventions during the biotransformation process. At optimized aeration and agitation conditions, a two-fold increase in inulinase activity could be noticed.

This study aimed to express an inulinase gene from the yeast Kluyveromyces marxianus (KmINU) in Aurantiochytrium sp. and realized one-step utilization of inulin resource for DHA production without any chemical pretreatment. An expression cassette with a length of 6052 bp for expressing the inulinase gene was constructed by a fast two-step PCR method and then was transferred into the Aurantiochytrium sp. cells. The Aurantiochytrium sp. recombinant T39 was selected with an inulinase activity up to 50.1 U/mL in 72 h. In a 5-l fed-batch fermentation, as high as 148.9 g/L of inulin was directly used within 120 h, and only 1.2 g/L of total sugar was left in the medium at the end of fermentation. The biomass of 51.4 g/L with a lipid content of 69.2% DCW and a DHA yield of 14.9 g/L was obtained.

To excavate the application of Jerusalem artichoke on poly(γ-glutamic acid) (γ-PGA) production, a γ-PGA producing strain Bacillus amyloliquefaciens NX-2S154 was obtained through atmospheric and room temperature plasma mutagenesis, which produced 14.83 ± 0.31 g/L of γ-PGA in batch fermentation with raw inulin extract. Simultaneous saccharification and fermentation (SSF) by adding commercial inulinase were further investigated for γ-PGA fermentation. Results showed SSF could eliminate the ineffective utilization of inulin while avoiding inhibition effect of high concentration substrate, which made γ-PGA concentration reach 18.54 ± 0.39 g/L with the process being shortened by 17%. Finally, an immobilized column for reducing inulinase cost was introduced to γ-PGA production. Repeated batch cultures showed the novel bioreactor exhibited higher stability and simplicity and gave average γ-PGA concentration and productivity of 19.40 ± 0.37 g/L and 0.27 ± 0.008 g/L/h, respectively. This work proposes a productive method for efficient γ-PGA production using Jerusalem artichoke feedstock.

Inulin is a readily available feedstock for cost-effective production of biochemicals. To date, several studies have explored the production of bioethanol, high-fructose syrup and fructooligosaccharide, but there are no studies regarding the production of d-lactic acid using inulin as a carbon source. In the present study, chicory-derived inulin was used for d-lactic acid biosynthesis by Lactobacillus bulgaricus CGMCC 1.6970. Compared with separate hydrolysis and fermentation processes, simultaneous saccharification and fermentation (SSF) has demonstrated the best performance of d-lactic acid production. Because it prevents fructose inhibition and promotes the complete hydrolysis of inulin, the highest d-lactic acid concentration (123.6 ± 0.9 g/L) with a yield of 97.9 % was obtained from 120 g/L inulin by SSF. Moreover, SSF by L. bulgaricus CGMCC 1.6970 offered another distinct advantage with respect to the higher optical purity of d-lactic acid (>99.9 %) and reduced number of residual sugars. The excellent performance of d-lactic acid production from inulin by SSF represents a high-yield method for d-lactic acid production from non-food grains.

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 +/- 0.03 U mL(-1)) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 +/- 0.03 U mL(-1) after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L(-1) h(-1)) and specific (119,025 U g(-1) h(-1)) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L(-1) h(-1) and specific productivity of 72 g g(-1) h(-1) FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.

The optimization of process parameters for high inulinase production by the marine yeast strain Cryptococcus aureus G7a in solid-state fermentation (SSF) was carried out using central composite design (CCD), one of the response surface methodologies (RSMs). We found that moisture, inoculation size, the amount ratio of wheat bran to rice husk, temperature and pH had great influence on inulinase production by strain G7a. Therefore, the CCD was used to evaluate the influence of the five factors on the inulinase production by strain G7a. Then, five levels of the five factors above were further optimized using the CCD. Finally, the optimal parameters obtained with the RSM were the initial moisture 61.5%, inoculum 2.75%, the amount ratio of wheat bran to rice husk 0.42, temperature 29 degrees C, pH 5.5. Under the optimized conditions, 420.9 U g(-1) of dry substrate of inulinase activity was reached in the solid-state fermentation culture of strain G7a within 120 h whereas the predicted maximum inulinase activity of 436.2 U g(-1) of inulinase activity of 436.2 U g(-1) of dry weight was derived from the RSM regression. This is the highest inulinase activity produced by the yeast strain reported so far. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.

The production of inulinase employing agroindustrial residues as the substrate is a good alternative to reduce production costs and to minimize the environmental impact of disposing these residues in the environment. This study focused on the use of a phenomenological model and an artificial neural network (ANN) to simulate the inulinase production during the batch cultivation of the yeast Kluyveromyces marxianus NRRL Y-7571, employing a medium containing agroindustrial residues such as molasses, corn steep liquor and yeast extract. It was concluded that due to the complexity of the medium composition it was rather difficult to use a phenomenological model with sufficient accuracy. For this reason, an alternative and more cost-effective methodology based on ANN was adopted. The predictive capacity of the ANN was superior to that of the phenomenological model, indicating that the neural network approach could be used as an alternative in the predictive modeling of complex batch cultivations.

This work was aimed at producing inulinase by solid-state fermentation of sugarcane bagasse, using factorial design to identify the effect of corn steep liquor (CSL) and soybean bran concentration, particle size of bagasse and size of inoculum. Maximum inulinase activity achieved was 250 U per g of dry substrate (gds) at 20% (w/w) of CSL, 5% (w/w) of soybean bran, 1 × 1010 cells mL−1 and particle size of bagasse in the range 9/32 mesh. The use of soybean bran decreased the time to reach maximum activity from 96 to 24 h and the maximum productivity achieved was 8.87 U gds−1 h−1. The maximum activity was obtained at pH 5.0 and 55.0°C. Within the investigated range, the enzyme extract was more thermostable at 50.0°C, showing a D-value of 123.1 h and deactivation energy of 343.9 kJ gmol−1. The extract showed highest stability from pH 4.5 to 4.8. Apparent K m and V max are 7.1 mM and 17.79 M min−1, respectively.

Inulin is a polyfructan that is abundant in plants such as Jerusalem artichoke, chicory and dahlia. Inulinase can easily hydrolyze inulin to fructose, which is consumed by microorganisms. Generally, Saccharomyces cerevisiae, an industrial workhorse strain for bioethanol production, is known for not having inulinase activity. The inulinase gene from Kluyveromyces marxianus (KmINU), with the ability of converting inulin to fructose, was introduced into S. cerevisiae D452-2. The inulinase gene was fused to three different types of promoter (GPD, PGK1, truncated HXT7) and secretory signal sequence (KmINU, MFα1, SUC2) to generate nine expression cassettes. The inulin fermentation performance of the nine transformants containing different promoter and signal sequence combinations for inulinase production were compared to select an optimized expression system for efficient inulin fermentation. Among the nine inulinase-producing transformants, the S. cerevisiae carrying the PGK1 promoter and MFα1 signal sequence (S. cerevisiae D452-2/p426PM) showed not only the highest specific KmINU activity, but also the best inulin fermentation capability. Finally, a batch fermentation of the selected S. cerevisiae D452-2/p426PM in a bioreactor with 188.2 g/L inulin was performed to produce 80.2 g/L ethanol with 0.43 g ethanol/g inulin of ethanol yield and 1.22 g/L h of ethanol productivity.

An experimental design was carried out to evaluate the effect of the concentrations of sodium alginate, glutaraldehyde and activated coal on the immobilization of inulinase from Kluyveromyces marxianus NRRL Y-7571. The experimental condition of 20 g/L of sodium alginate, 50 mL/L of glutaraldehyde and 30 g/L of activated coal led to the highest specific activity (2,063.5 U/mg of protein), corresponding to an enhancement of about 26 times compared to the activity of the free enzyme (79.1 U/mg of protein). The effect of pH and temperature on the immobilized enzyme activity was also evaluated, showing optimal activities at pH of 5.5 and 55 °C. The study of storage of immobilized inulinase in different temperatures showed that the extract kept its initial activity after 43 days of storage at 40 and 50 °C and after 138 days of storage either at 4 or 25 °C.

The present work aimed to study the viability of the use of sugarcane molasses and corn steep liquor (CSL) in a sequential inulinase production performing an up-stream pretreatment of these agroindustrial residues. A sequential strategy was used applying three central composite rotatable designs (CCRDs) to optimise medium composition, followed by a down-stream step. The medium containing 150 g L(-1) molasses, 50 g L(-1) CSL and 6 g L(-1) yeast extract, yielded a maximum inulinase production of 1,294 +/- 7 U mL(-1), after 72 h of fermentation. A down-stream evaluation was carried out using an expanded bed of Streamline DAE resin (Pharmacia), with and without the up-stream treatment. The results showed that the enzyme could not be recovered from the non-pretreated medium, whereas a yield of 91% was obtained in the adsorption stage from the medium prepared with the up-stream treatment, showing the viability of producing the enzyme inulinase from agroindustrial residues using the integrated process.

A new method for determination of the optimum temperature and activation energies based on an idea of the average rate of enzymatic reaction has been developed. A mathematical model describing the effect of temperature on a dimensionless activity for enzyme deactivation following the first-order kinetics has been derived. The necessary condition for existence of the function extreme of the optimal temperature has been applied in the model. The developed method has been verified using the experimental data for inulinase from Kluyveromyces marxianus.