Direct Production of (R)-3-Hydroxybutyric Acid of High Optical Purity by Halomonas sp. OITC1261 Under Aerobic conditions.

The chiral compound (R)-3-hydroxybutyric acid (R-3HB) has some important physiological functions in the human body and can also be applied as a functional material for industrial products. A novel Halomonas sp. OITC1261 isolated from a coastal sediment is selected for efficient production of R-3HB. This strain secreted R-3HB of high optical purity into the extracellular medium while maintaining poly(3-hydroxybutyric acid) (PHB) inside the microbial cell under aerobic conditions. There is a possibility that R-3HB is generated independently of PHB. Under aerobic cultivation, 58 g L-1 (0.65 g L-1 h-1 ) of R-3HB was produced simultaneously with 27 g L-1 (0.30 g L-1 h-1 ) of PHB. This direct production method of R-3HB contributes to the improvement of mass production technology.

Isolation of alkaliphilic bacteria for production of high optically pure L-(+)-lactic acid.

Lactic acid bacteria that grow under alkaline conditions (pH 10) were isolated from various sources in Okinawa (Japan). These alkali-tolerant and alkaliphilic bacteria were classified as follows: Microbacterium sp. (1 strain), Enterococcus spp. (9 strains), Alkalibacterium spp. (3 strains), Exiguobacterium spp. (5 strains), Oceanobacillus spp. (3 strains) and Bacillus spp. (7 strains) by 16S rRNA gene sequencing. By fermentation, many strains were able to convert glucose into mainly L-(+)-lactic acid of high optical purity in alkaline broth. This result indicated that valuable L-(+)-lactic acid-producing bacteria could be isolated efficiently by screening under alkaline conditions. Six strains were selected and their ability to produce lactic acid at different initial pH was compared. Enterococcus casseliflavus strain 79w3 gave the highest lactic acid concentration. Lactic acid concentration and productivity were 103 g L(-1) (optical purity of 99.5% as L-isomer) and 2.2 g L(-1) h(-1), respectively when 129 g L(-1) of glucose was used by batch fermentation.

Efficient production of D-(-)-lactic acid from broken rice by Lactobacillus delbrueckii using Ca(OH)2 as a neutralizing agent.

Effects of Ca(OH)(2), NH(4)OH, and NaOH as neutralizing agents for efficient recovery of lactic acid was investigated. Lactic acid was produced from broken rice in a simultaneous saccharification and fermentation (SSF) process with Lactobacillus delbrueckii. Consumption of glucose (from broken rice) by the cells and the cell growth were the best with Ca(OH)(2) among the three neutralizing agents used. Maximum productivities of lactic acid reached with Ca(OH)(2), NH(4)OH, and NaOH were 3.59 g l(-1) h(-1), 1.51 g 1(-1) h(-1), and 1.40 g l(-1) h(-1), respectively. Ca(OH)(2) reduced the lactate molarity of the fermentation broth, and thus resulted in the highest lactic acid productivity. Furthermore, it was apparently clear that divalent cation (Ca(2+)) was more effective in neutralizing the cultures compared to monovalent (Na(+) and NH(3)(+)) cations.

Production of (R)-3-hydroxybutyric acid by fermentation and bioconversion processes with Azohydromonas lata.

Feasibility of producing (R)-3-hydroxybutyric acid ((R)-3-HB) using wild type Azohydromonas lata and its mutants (derived by UV mutation) was investigated. A. lata mutant (M5) produced 780 m g/l in the culture broth when sucrose was used as the carbon source. M5 was further studied in terms of its specificity with various bioconversion substrates for production of (R)-3-HB. (R)-3-HB concentration produced in the culture broth by M5 mutant was 2.7-fold higher than that of the wild type strain when sucrose (3% w/v) and (R,S)-1,3-butanediol (3% v/v) were used as carbon source and bioconversion substrate, respectively. Bioconversion of resting cells (M5) with glucose (1% v/w), ethylacetoacetate (2% v/v), and (R,S)-1,3-butanediol (3% v/v), resulted in (R)-3-HB concentrations of 6.5 g/l, 7.3g/l and 8.7 g/l, respectively.

Fermentative production of L: -(+)-lactic acid by an alkaliphilic marine microorganism.

Of six strains of lactic acid-producing alkaliphilic microorganisms, Halolactibacillus halophilus was most efficient. It produced the highest concentration and yield of lactic acid, with minimal amounts of acetic and formic acid when sucrose and glucose were used as substrate. Mannose and xylose were poorly utilized. In batch fermentation at 30°C, pH 9 with 4 and 8% (w/v) sucrose, lactic acid was produced at 37.7 and 65.8 g l(-1), with yields of 95 and 83%, respectively. Likewise, when 4 and 8% (w/v) glucose were used, 33.4 and 59.6 g lactic acid l(-1) were produced with 85 and 76% yields, respectively. L: -(+)-lactic acid had an optical purity of 98.8% (from sucrose) and 98.3% (from glucose).

Biodegradability of plastics.

Plastic is a broad name given to different polymers with high molecular weight, which can be degraded by various processes. However, considering their abundance in the environment and their specificity in attacking plastics, biodegradation of plastics by microorganisms and enzymes seems to be the most effective process. When plastics are used as substrates for microorganisms, evaluation of their biodegradability should not only be based on their chemical structure, but also on their physical properties (melting point, glass transition temperature, crystallinity, storage modulus etc.). In this review, microbial and enzymatic biodegradation of plastics and some factors that affect their biodegradability are discussed.

Application of bipolar electrodialysis on recovery of free lactic acid after simultaneous saccharification and fermentation of cassava starch.

The efficiency of bipolar electrodialysis (BED) for the recovery of lactic acid from fermentation broth was evaluated. Three systems of BED (bipolar-anion, bipolar-cation and bipolar-anion-cation) at fixed voltage (20 V) were compared using a model solution of ammonium lactate (100 g l(-1)). Results showed that bipolar-anion (BED-anion) was the most beneficial in terms of lactate flux, current efficiency, energy consumption and recovery ratio. Consequently, BED-anion was used to purify lactic acid from fermentation broth which had been pre-treated with mono-polar electrodialysis (MED). The final lactic acid concentration and lactate flux obtained were 144 g l(-1) and 393 g m(-2) h(-1), respectively. Using the two-step process (MED and BED-anion) the concentration of fermentation broth was increased by 33% and the total energy consumption was 2.76 kW h kg(-1).

Alkalibacterium indicireducens sp. nov., an obligate alkaliphile that reduces indigo dye.

Indigo-reducing, obligately alkaliphilic strains A11T, F11 and F12 were isolated from indigo fermentation liquor obtained from Tokushima Prefecture, Shikoku, Japan. The isolates grew at pH 9.0-12.3, but not at pH 7.0-8.0. The optimum pH range for growth was 9.5-11.5. They were Gram-negative, facultatively anaerobic, rod-shaped strains with peritrichous flagella. The isolates grew in 0-14 % (w/v) NaCl, with optimum growth at 1-11 %. They grew at temperatures of 15-35 degrees C with optimum growth at around 20-30 degrees C. dl-Lactate was the major end product from d-glucose. No quinones were detected. The peptidoglycan type was A4 alpha, l-Lys (l-Orn)-d-Asp. The major cellular fatty acids were C16 : 0, C16 : 17c and C18 : 19c. The DNA G+C contents were 47.0-47.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence data indicated that the isolates belong to the genus Alkalibacterium. DNA-DNA hybridization revealed low relatedness values between the isolates and the three phylogenetically most closely related species, Alkalibacterium olivapovliticus, Alkalibacterium psychrotolerans and Alkalibacterium iburiense (<41 %). On the basis of phenotypic characteristics, including hydrolysis of cellulose and fermentation of carbohydrates, and chemotaxonomic characteristics, phylogenetic data and DNA-DNA relatedness data, it is concluded that the isolates merit classification as representatives of a novel species of the genus Alkalibacterium, for which the name Alkalibacterium indicireducens sp. nov. is proposed. The type strain of this species is A11T (=JCM 14232T=NCIMB 14253T).

Production of D-lactic acid from sugarcane molasses, sugarcane juice and sugar beet juice by Lactobacillus delbrueckii.

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40 degrees C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l(-1)) and sugarcane juice (133 g total sugar l(-1)) was 107 g l(-1) and 120 g l(-1), respectively. With 10% (w/v) sugar beet juice (105 g total sugar l(-1)), 84 g lactic acid l(-1) was produced. The optical purities of D: -lactic acid from the feedstocks ranged from 97.2 to 98.3%.

Biotechnological production of (R)-3-hydroxybutyric acid monomer.

The escalating problems regarding the treatment of plastic waste materials have led to development of biodegradable plastics. At present, a number of aliphatic polyesters; such as poly[(R)-3-hydroxybutyrate] (PHB), poly(l-lactide), polycaplolactone, poly(ethylene succinate) and poly(butylene succinate) have been developed. Among these aliphatic polyesters, PHB is one of the most attractive since it can undergo biodegradation at various environmental conditions and has properties similar to polypropylene. Although much effort has been made to produce PHB and its copolyesters from renewable resources or through microbial processes, their commercialization and widespread application are still not economically attractive compared to conventional non-biodegradable plastic. Moreover, wide application of PHB and its copolyesters as biodegradable plastic have not only been limited by the cost of production but also by their stinky smell during industrial processing. However, (R)-3-hydroxybutyric acid, a monomer of PHB has wide industrial and medical applications. (R)-3-hydroxybutyric acid can also serve as chiral precursor for synthesis of pure biodegradable PHB and its copolyesters. A number of options are available for production of (R)-3-hydroxybutyric acid. This review discusses each of these options to assess the alternatives that exist for production of pure biodegradable PHB and its copolyesters with good properties.

Enzymatic synthesis of arbutin undecylenic acid ester and its inhibitory effect on melanin synthesis.

Transesterification of arbutin and undecylenic acid vinyl ester was catalyzed by alkaline protease, Bioprase, in dimethylformamide to get arbutin derivative having undecylenic acid at 6-position of glucose moiety, 6-O-undecylenoyl p-hydroxyphenyl beta-D-glucopyranoside. The reaction rate increased with increase of arbutin concentration, and when its concentration was 0.9 M, the conversion rate was more than 90% under addition of 2 M undecylenic acid vinyl ester. The obtained arbutin ester significantly suppressed melanin production in murine B16 melanoma cells.

Biodegradability and biodegradation of poly(lactide).

Poly(lactide) (PLA) has been developed and made commercially available in recent years. One of the major tasks to be taken before the widespread application of PLA is the fundamental understanding of its biodegradation mechanisms. This paper provides a short overview on the biodegradability and biodegradation of PLA. Emphasis is focused mainly on microbial and enzymatic degradation. Most of the PLA-degrading microorganisms phylogenetically belong to the family of Pseudonocardiaceae and related genera such as Amycolatopsis, Lentzea, Kibdelosporangium, Streptoalloteichus, and Saccharothrix. Several proteinous materials such as silk fibroin, elastin, gelatin, and some peptides and amino acids were found to stimulate the production of enzymes from PLA-degrading microorganisms. In addition to proteinase K from Tritirachium album, subtilisin, a microbial serine protease and some mammalian serine proteases such as alpha-chymotrypsin, trypsin, and elastase could also degrade PLA.

Lactic acid production from sugar-cane juice by a newly isolated Lactobacillus sp.

A newly isolated sucrose-tolerant, lactic acid bacterium, Lactobacillus sp. strain FCP2, was grown on sugar-cane juice (125 g sucrose l(-1), 8 g glucose l(-1) and 6 g fructose l(-1)) for 5 days and produced 104 g lactic acid l(-1) with 90% yield. A higher yield (96%) and productivity (2.8 g l(-1 )h(-1)) were obtained when strain FCP2 was cultured on 3% w/v (25 g sucrose l(-1), 2 g glucose l(-1) and 1 g fructose l(-1)) sugar-cane juice for 10 h. Various cheap nitrogen sources such as silk worm larvae, beer yeast autolysate and shrimp wastes were also used as a substitute to yeast extract.

A novel PHB depolymerase from a thermophilic Streptomyces sp.

A novel PHB depolymerase from a thermophilic Streptomyces sp. MG was purified to homogeneity by hydrophobic interaction chromatography and gel filtration. The molecular mass of the purified enzyme was 43 kDa as determined by size exclusion chromatography and 41 kDa by SDS-PAGE. The optimum pH and temperature were 8.5 and 60 degrees C respectively. The enzyme was stable at 50 degrees C and from pH 6.5-8.5. The enzyme hydrolyzed not only bacterial polyesters, i.e. poly(3-hydroxybutyric acid and poly(3-hydroxybutyrate-co-3-hydroxyvalerate), but also synthetic, aliphatic polyesters such as polypropiolactone, poly(ethylene adipate) and poly(ethylene succinate).

Production of poly(L-lactide)-degrading enzyme by Amycolatopsis orientalis for biological recycling of poly(L-lactide).

Efficient production of poly(L-lactide)(PLA)-degrading enzyme was achieved by addition of 0.1% (w/v) silk fibroin powder into a liquid culture medium of an actinomycete, Amycolatopsis orientalis, without other complex nitrogen sources, such as yeast extract and peptone. Scaled-up production of the enzyme in a 5-l jar fermenter showed the possibility of producing this enzyme on an industrial scale at low production cost. The extracellular PLA-degrading enzyme showed potent degrading activity, which is effective for biological recycling of PLA, i.e., 2,000 mg/l of PLA powder was completely degraded within 8 h at 40 degrees C using 20 mg/l purified enzyme. An optically active L-lactic acid with 600 mg/l was obtained as degradation product of PLA without undesirable racemization.

Hydrolysis of polyesters by serine proteases.

The substrate specificity of alpha-chymotrypsin and other serine proteases, trypsin, elastase, proteinase K and subtilisin, towards hydrolysis of various polyesters was examined using poly(L-lactide) (PLA), poly(beta-hydroxybutyrate) (PHB), poly(ethylene succinate) (PES), poly(ethylene adipate) (PEA), poly(butylene succinate) (PBS), poly(butylene succinate-co-adipate) (PBS/A), poly[oligo(tetramethylene succinate)-co-(tetramethylane carbonate)] (PBS/C), and poly(epsilon-caprolactone) (PCL). alpha-Chymotrypsin could degrade PLA and PEA with a lower activity on PBS/A. Proteinase K and subtilisin degraded almost all substrates other than PHB. Trypsin and elastase had similar substrate specificities to alpha-chymotrypsin.

A new method for the evaluation of biodegradable plastic using coated cellulose paper.

A highly sensitive analytical method for evaluation of poly(L-lactide) (PLA), poly(epsilon-caprolactone) (PCL), poly(beta-hydroxybutyrate) (PHB), and poly(butylene succinate) (PBS) degradability was developed using coated cellulose paper, prepared by penetration and adhesion of these plastics into/onto the cellulose paper. Enzymatic degradability of the obtained plastic coated papers was evaluated using various commercial proteases and lipases. PLA coated paper was highly susceptible to subtilisin and mammalian enzymes, alpha-chymotrypsin, elastase and trypsin. To our knowledge, this is the first report on the degradation of PLA coated paper using subtilisin and mammalian enzymes. Almost all lipase preparations degraded PCL and PHB coated papers but not PBS coated paper. The biodegradability of plastic coated paper was greater than that of plastic powder. The penetration of plastic into cellulose paper by coating improved the plastic degradability, and can be regulated easily.

Enzymatic transesterification of purine nucleoside having a low solubility in organic medium.

Enzymatic transesterification of guanosine having low solubility against organic solvent was examined. For the transesterification between guanosine and divinyl adipate catalyzed by alkaline protease from Bacillus (Bioprase), DMSO was added to DMF to increase the solublility of the nucleoside, and the conversion rate of guanosine to the vinyl guanosine ester was less than 30%. To overcome the reversible inactivation of enzyme by hydrophilic organic solvents, the reaction was carried out with 10% (v/v) water. The transesterification reaction was effectively catalyzed in DMF/DMSO in the presence of water and the conversion rate increased ca. 70% after 7 d reaction. The result shows that the water effect of Bioprase would be a useful method for the synthesis of low solublility nucleoside esters.

Degradation of microbial polyesters.

Microbial polyhydroxyalkanoates (PHAs), one of the largest groups of thermoplastic polyesters are receiving much attention as biodegradable substitutes for non-degradable plastics. Poly(D-3-hydroxybutyrate) (PHB) is the most ubiquitous and most intensively studied PHA. Microorganisms degrading these polyesters are widely distributed in various environments. Although various PHB-degrading microorganisms and PHB depolymerases have been studied and characterized, there are still many groups of microorganisms and enzymes with varying properties awaiting various applications. Distributions of PHB-degrading microorganisms, factors affecting the biodegradability of PHB, and microbial and enzymatic degradation of PHB are discussed in this review. We also propose an application of a new isolated, thermophilic PHB-degrading microorganism, Streptomyces strain MG, for producing pure monomers of PHA and useful chemicals, including D-3-hydroxycarboxylic acids such as D-3-hydroxybutyric acid, by enzymatic degradation of PHB.

Biodegradation of poly(L-lactide).

The biodegradation of poly(L-lactide) (PLA) is reviewed. The important role of actinomycetes in PLA degradation is emphasized. These PLA-degrading actinomycetes belong phylogenetically to the Pseudonocardiaceae family and related genera, including Amycolatopsis, Lentzea, Streptoalloteichus, Kibdelosporangium and Saccharothrix. A PLA-degrading enzyme purified from an isolated Amycolatopsis strain-41 has substrate specificity on PLA higher than proteinase K. The application of these strains and their enzymes can be effectively used for biological treatment of plastic wastes containing PLA.