Production of high crystallinity type-I cellulose from Komagataeibacter hansenii JR-02 isolated from Kombucha tea.

In this study, a bacterial cellulose (BC) producing strain was isolated from Kombucha tea and identified as Komagataeibacter hansenii JR-02 by morphological, physiological, and biochemical characterization and 16S rRNA sequence. Then, the media components and culture conditions for BC production were optimized. Result showed that the highest BC yield was 3.14 ± 0.22 and 8.36 ± 0.19 g/L after fermentation for 7 days under shaking and static cultivation, respectively. Moreover, it was interesting that JR-02 could produce BC in nitrogen-free medium with the highest yield of 0.76 ± 0.06 g/L/7days, and the possible nitrogen fixation gene nifH was cloned from its genomic DNA. The BC produced by JR-02 was type-I cellulose with high crystallinity and thermodynamic stability, which was revealed from Fourier transform infrared spectroscopy, X-ray diffraction, and thermogravimetric analysis methods. The crystallinity of static and shaking cultured BC were 91.76% and 90.69%, respectively. The maximum rate of weight loss of static and shaking BC occurred at temperature of approximately 373.1 °C and 369.1 °C, respectively. Overall, these results indicated that K. hansenii JR-02 had great potential to produce high crystallinity type-I BC in manufacture.

Poly(malic acid) production from liquefied corn starch by simultaneous saccharification and fermentation with a novel isolated Aureobasidium pullulans GXL-1 strain and its techno-economic analysis.

In this study, a novel Aureobasidium pullulans GXL-1 strain without melanin secretion was isolated for efficient polymalic acid (PMA) production. The PMA produced by GXL-1 was characterized, and its molecular mass was determined to be 1.621 kDa by gel permeation chromatography. Liquefied corn starch was shown to replace glucose for PMA production by GXL-1 through simultaneous saccharification and fermentation. The PMA titer obtained from batch fermentation was up to 49.0 ± 1.6 g/L in a 10 L fermentor, and the PMA yield and productivity obtained from repeated-batch fermentation were up to 0.50 g/g and 0.34 g/L·h, respectively. Furthermore, process design and techno-economic analysis were performed at an annual output level of 5000 metric tons by SuperPro Designer. Results showed that the production cost of $2.046/kg and payback period of 6.9 years were achieved by repeated-batch fermentation; this provides an economically feasible strategy for industrial-scale production of PMA.

Analysis of the L-malate biosynthesis pathway involved in poly(ß-L-malic acid) production in Aureobasidium melanogenum GXZ-6 by addition of metabolic intermediates and inhibitors.

Poly(ß-L-malic acid) (PMA) is a promising polyester formed from L-malate in microbial cells. Malate biosynthesis is crucial for PMA production. Previous studies have shown that the non-oxidative pathway or oxidative pathway (TCA cycle) is the main biosynthetic pathway of malate in most of PMA-producing strains, while the glyoxylate cycle is only a supplementary pathway. In this study, we investigated the effect of exogenous metabolic intermediates and inhibitors of the malate biosynthetic pathway on PMA production by Aureobasidium melanogenum GXZ-6. The results showed that PMA production was stimulated by maleic acid (a fumarase inhibitor) and sodium malonate (a succinate dehydrogenase inhibitor) but inhibited by succinic acid and fumaric acid. This indicated that the TCA cycle might not be the only biosynthetic pathway of malate. In addition, the PMA titer increased by 18.1% upon the addition of glyoxylic acid after 72 h of fermentation, but the PMA titer decreased by 7.5% when itaconic acid (an isocitrate lyase inhibitor) was added, which indicated that malate for PMA production was synthesized significantly via the glyoxylate cycle rather than the TCA cycle. Furthermore, in vitro enzyme activities of the TCA and glyoxylate cycles suggested that the glyoxylate cycle significantly contributed to the PMA production, which is contradictory to what has been reported previously in other PMA-producing A. pullulans.

Efficient Production of Polymalic Acid by a Novel Isolated Aureobasidium pullulans Using Metabolic Intermediates and Inhibitors.

Polymalic acid (PMA) is a linear anionic polyester composed of L-malic acid monomers, which have potential applications as drug carriers, surgical suture, and biodegradable plastics. In this study, a novel strain of Aureobasidium pullulans var. melanogenum GXZ-6 was isolated and identified according to the morphological observation and deoxyribonucleic acid internal-transcribed spacer sequence analysis, and the product of PMA was characterized by FT-IR, 13C-NMR, and 1H-NMR spectra. The PMA titer of GXZ-6 reached 62.56 ± 1.18 g L-1 with productivity of 0.35 g L-1 h-1 using optimized medium with addition of metabolic intermediates (citrate and malate) and inhibitor (malonate) by batch fermentation in a 10-L fermentor. Besides that the malate for PMA synthesis in GXZ-6 might mainly come from the glyoxylate cycle, based on results, citrate, malate, malonate, and maleate increased while succinate and fumarate inhibited the production of PMA, which was different from that of other A. pullulans. This study provided a potential strain and a simple metabolic control strategy for high-titer production of PMA and shared novel information on the biosynthesis pathway of PMA in A. pullulans.

Development and benefit evaluation of fermentation strategies for poly(malic acid) production from malt syrup by Aureobasidium melanogenum GXZ-6.

Malt syrup, as a low-cost substrate without any pretreatment, was proved to be able to replace maltose for ploymalic acid (PMA) production by Aureobasidium melanogenum GXZ-6. The PMA titer of 55.53 ±â€¯1.72 g/L was obtained by batch fermentation in a 10-L fermentor with addition of malate, citrate and sodium malonate. Then, a higher PMA titer of 124.07 ±â€¯2.28 g/L was obtained in fed-batch fermentation, which increased by 123.43% than that from batch fermentation. Moreover, repeated-batch fermentation with three batches gave a PMA titer of 64.06 g/L on average with a higher yield of 0.81 g/g and productivity of 0.56 g/L·h. Fermentation process and economics analysis were performed by SuperPro Designer for a 2000 metric tons plant. Results showed that PMA production cost was as low as $ 1.716/kg by fed-batch fermentation, which provides an economical strategy for large-scale PMA production.

Purification and characterization of a cellulase-free, thermostable endo-xylanase from Streptomyces griseorubens LH-3 and its use in biobleaching on eucalyptus kraft pulp.

Xylanase is an important enzyme involved in degrading xylan. In this study, an extracellular cellulase-free, thermostable endo-xylanase which was produced by Streptomyces griseorubens LH-3 with bagasse semi-cellulose as a carbon source was purified and characterized. The xylanase was purified 4-fold with a recovery yield of 21.6% by precipitation with 25-55% (NH4)2SO4, Mono Q ion exchange chromatography and sephacryl S-200 HR gel filtration chromatography. It appeared as a monomeric protein on SDS-PAGE gel and had an apparent molecular weight of 45.5 kDa with specific activity of 434 IU/mg. Using birchwood xylan as substrate, the maximum velocity (Vmax) and Michaelis-Menten constant (Km) were found to be 1.44 mg/ml and 2.05 µmol/min mg, respectively. The purified xylanase was active at pH 4.0-8.0 with an optimum pH of 5.0. It was stable at temperatures between 30°C and 50°C, exhibiting maximum activity at 60°C. Hg2+ and Al3+ inhibited the enzyme activity significantly. Enzymatic product analysis indicated that the enzyme was an endo-xylanase, whose hydrolysis products were mainly a series of short-chain xylooligosaccharides. Furthermore, it was used for biobleaching of eucalyptus kraft pulp, and results showed that this purified xylanase increased the brightness of the pulp by 14.5% and reduced the kappa number by 24.5%. All these industrially relevant characteristics made it had potential application in the pulp and paper industry as a biobleaching agent.