Utilization of volatile fatty acids from the anaerobic digestion liquor of sewage sludge for 5-aminolevulinic acid production by photosynthetic bacteria

Using volatile fatty acids (VFA) from the anaerobic digestion liquor of sewage sludge, up to 9.2 mm 5-aminolevulinic acid (ALA) could be produced by Rhodobacter sphaeroides under anaerobic-light (5 kLux) conditions with repeated addition of levulinic acid (LA) and glycine and using a large inoculum (approx. 2 g/l of cells, initially from glutamate/malate medium). As the VFA medium also contained organic nitrogen sources such as glutamic acid, the cells were later grown up to about 2 g/l in the VFA medium instead of the glutamate/malate medium. ALA production was then again promoted by adding LA and glycine. Using this improved method, up to 9.3 mm ALA was produced by feeding propionate and acetate together with LA and glycine, indicating that VFA medium formed from sewage sludge could be useful for ALA production.     

Production of extracellular 5-aminolevulinic acid byClostridium thermoaceticum grown in minimal medium

Summary A growth associated formation of extracellular 5-aminolevulinic acid (ALA) was found in the homoacetogenesis of glucose byClostridium thermoaceticum grown in minimal defined medium. The growth and ALA production was enhanced by L-cysteine HCl both in complex medium (UM) and minimal defined medium (MDM). The amount of ALA produced extracellularly in MDM wasca. 15 mg/L after 90-h anaerobic cultivation (cell-mass: 1.5 g/l; glucose consumed: 20 g/l).     

Extracellular 5-aminolevulinic acid production by Escherichia coli containing the Rhodopseudomonas palustris KUGB306 hemA gene

The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'- phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).     

Coproporphyrin production by Rhodobacter sphaeroides under aerobic in the dark and dissolved-oxygen-controlled conditions

Porphyrin production under aerobic in the dark condition was carried out using the photosynthetic bacterium, Rhodobacter sphaeroides IFO12203 and its mutant, CR 386 which can produce 5-aminolevulinic acid (ALA) under aerobic in the dark conditions. IFO12203 produced about 1.0 mg/l of porphyrin even if 2.0 mg of ALA/l was added to the glucose–glutamate–yeast extract (GGY2) medium. However, CR 386 produced 15.0 mg/l of porphyrin after 55 h culture with the addition of 2.0 g of ALA/l and sufficient oxygen supply (dissolved oxygen, DO > 7.0 mg/l). The porphyrin produced by CR 386 consisted only of coproporphyrin III. Under conditions of strict DO control (DO = 2.0 ± 0.2 mg/l), the maximum porphyrin production attained 56.3 mg/l. Low DO (1.0 ± 0.2 mg/l) and high DO control (3.0 ± 0.2 mg/l) did not enhance porphyrin production. It is suggested that oxygen supply seems to control the step(s) of porphyrin biosynthesis of CR 386 in the stages after ALA synthase in the Shemin pathway.     

Enhancement of 5-aminolevulinate production with recombinant Escherichia coli using batch and fed-batch culture system

5-Aminolevulinate (ALA) production with recombinant Escherichia coli Rosetta (DE3)/pET28a(+)-hemA was studied. In batch fermentation, the addition of glucose and glycine was effective to improve ALA production. Then the fed-batch fermentation was conducted with continuous feeding of precursors. When the concentrations of succinic acid and glycine were 7.0 g/l and 4.0 g/l, respectively, in the feeding, the ALA yield reached 4.1g/l. But the molar yield (ALA/glycine) was decreased in the fed-batch fermentation compared to batch fermentation. And it was found that the pH control during fed-batch cultivation was very important for the cell growth and ALA production. A two-stage pH value controlling strategy was suggested, in which, the pH value in the first 6h was regulated at pH 5.9, after then at pH 6.2, and the ALA yield was as high as 6.6g/l via fed-batch fermentation.     

Identification and evaluation of ω-3 fatty acid desaturase genes for hyperfortifying α-linolenic acid in transgenic rice seed

α-Linolenic acid (ALA) deficiency and a skewed of ω6:ω3 fatty acid ratio in the diet are a major explanation for the prevalence of cardiovascular diseases and inflammatory/autoimmune diseases. There is a need to enhance the ALA content and to reduce the ratio of linoleic acid (LA) to ALA. Six ω-3 (Δ-15) fatty acid desaturase (FAD) genes were cloned from rice and soybean. The subcellular localizations of the proteins were identified. The FAD genes were introduced into rice under the control of an endosperm-specific promoter, GluC, or a Ubi-1 promoter to evaluate their potential in increasing the ALA content in seeds. The ALA contents in the seeds of endoplasmic reticulum (ER)-localized GmFAD3-1 and OsFAD3 overexpression lines increased from 0.36 mg g?1 to 8.57 mg g?1 and 10.06 mg g?1, respectively, which was 23.8- and 27.9-fold higher than that of non-transformants. The trait of high ALA content was stably inheritable over three generations. Homologous OsFAD3 is more active than GmFAD3-1 in catalysing LA conversion to ALA in rice seeds. Overexpression of ER-localized GmFAD3-2/3 and chloroplast-localized OsFAD7/8 had less effect on increasing the ALA content in rice seeds. The GluC promoter is advantageous compared with Ubi-1 in this experimental system. The enhanced ALA was preferentially located at the sn-2 position in triacylglycerols. A meal-size portion of high ALA rice would meet >80% of the daily adult ALA requirement. The ALA-rich rice could be expected to ameliorate much of the global dietary ALA deficiency.     

Screening and characteristics of a butanol-tolerant strain and butanol production from enzymatic hydrolysate of NaOH-pretreated corn stover

As a gasoline substitute, butanol has advantages over traditional fuel ethanol in terms of energy density and hydroscopicity. However, solvent production appeared limited by butanol toxicity. The strain of Clostridium acetobutylicum was subjected to mutation by mutagen of N-methyl-N'-nitro-N-nitrosoguanidine for 0.5?h. Screening of mutants was done according to the individual resistance to butanol. A selected butanol-resistant mutant, strain 206, produced 50?% higher solvent concentrations than the wild-type strain when 60?g glucose/l was employed as substrate. The strain was also able to produce solvents of 23.47?g/l in 80?g/l glucose P2 medium after 70?h fermentation, including 5.41?g acetone/l, 15.05?g butanol/l and 3.02?g ethanol/l, resulting in an ABE yield and productivity of 0.32?g/g and 0.34?g/(l?h). Subsequently, Acetone-butanol-ethanol (ABE) production from enzymatic hydrolysate of NaOH-pretreated corn stover was investigated in this study. An ABE yield of 0.41 and a productivity of 0.21?g/(l?h) was obtained, compared to the yield of 0.33 and the productivity of 0.20?g/(l?h) in the control medium containing 52.47 mixed sugars. However, it is important to note that although strain 206 was able to utilize all the glucose rapidly in the hydrolysate, only 32.9?% xylose in the hydrolysate was used after fermentation stopped compared to 91.4?% xylose in the control medium. Strain 206 was shown to be a robust strain for ABE production from lignocellulosic materials and has a great potential for industrial application.     

Influence of isolation technique of half-anthers and of initiation culture medium on callus induction and regeneration in Anthurium andreanum

Optimization of the isolation technique and initiation culture medium are two critical aspects that can determine the success of anthurium half-anther culture. Both aspects in half-anther culture of Anthurium andreanum Linden ex André cv. ??Tropical?? were studied and successfully improved. Untreated half-anthers, when cultured abaxial side down on medium, was the most suitable means of inducing callus. Callus formation was further improved by culturing half-anthers adaxial side down on Winarto-Teixeira (WT) medium (Winarto et al. in Plant Growth Regul 65:513?C529, 2011b) supplemented with 0.01?mg/l ??-naphthaleneacetic acid (NAA), 1.0?mg/l 6-benzyladenine (BA) and 0.5?mg/l thidiazuron (TDZ). Gelrite enhanced callus formation (compared to agar) when the concentration was reduced from 2.0 to 1.5?g/l on WT medium. Application of 0.5?mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) in WT medium increased callus formation. Most callus formed when half-anthers were cultured adaxial-side down on WT medium supplemented with 0.5?mg/l 2,4-D in combination with 0.01?mg/l NAA, 1.0?mg/l BA and 0.5?mg/l TDZ using 1.5?g/l Gelrite. This ideal medium induced the growth of half-anthers, with as much as 38?% of half-anthers producing callus, or, on average, 3.4 half-anthers/treatment. Callus derived from this optimized protocol regenerated easily and could be multiplied on New Winarto-Teixeira medium (NWT) (Winarto et al. in Plant Growth Regul 65:513?C529, 2011b) containing 0.25?mg/l 2,4-D, 0.02?mg/l NAA, 0.75?mg/l BA, 1.5?mg/l TDZ and 2.0?g/l Gelrite. Shoots rooted well on hormone-free NWT medium with 2.0?g/l Gelrite. The plantlets could be easily acclimatized in a substrate of raw rice husk, burned-rice husk and organic manure (1:1:1, v/v/v) with a high survival (100?%) ex vitro in a greenhouse. The results of this study would benefit half-anther culture of other Anthurium cultivars, particularly at the initial stage of callus induction.     

Valorisation of chicken feathers for xanthan gum production using Xanthomonas campestris MO-03

Xanthan gum is an important commercial polysaccharide produced by Xanthomonas species. In this study, xanthan production was investigated using a local isolate of Xanthomonas campestris MO-03 in medium containing various concentrations of chicken feather peptone (CFP) as an enhancer substrate. CFP was produced with a chemical process and its chemical composition was determined. The addition of CFP (1–8?g/l) increased the conversion of sugar to xanthan gum in comparison with the control medium, which did not contain additional supplements. The highest xanthan production (24.45?g/l) was found at the 6?g/l CFP containing control medium in 54?h. This value was 1.73 fold higher than that of control medium (14.12?g/l). Moreover, addition of CFP improved the composition of xanthan gum; the pyruvate content of xanthan was 3.86% (w/w), higher than that of the control (2.2%, w/w). The xanthan gum yield was also influenced by the type of organic nitrogen sources. As a conclusion, CFP was found to be a suitable substrate for xanthan gum production.     

Optimization of recombinant aminolevulinate synthase production in<Emphasis Type="Italic"> Escherichia coli</Emphasis> using factorial design

The production of recombinant Rhodobacter sphaeroides aminolevulinate (ALA) synthase was optimized in two strains of Escherichia coli: the wild-type strain MG1655, and a ptsG mutant AFP111. The effects of initial succinate, glucose and isopropyl--d-thiogalactopyranoside (IPTG) concentrations and the time of induction on enzyme activity were studied. One-way analysis was used to approximate the optimal ranges for these factors, followed by a full factorial design to quantify the effects of each factor and the interactions between the factors. Initial succinate, glucose, and IPTG concentration were observed to be the key factors affecting ALA synthase activity with the optimal levels determined to be above 6 g/l succinate, 0 g/l glucose, and 0.10 mM IPTG. ALA synthase activity was generally lower with AFP111 than with MG1655, and the effect of these three key factors was also lower with AFP111 than with MG1655. Based on the full factorial design results, a fermentation was completed that yielded 296 mU/mg protein with a final ALA concentration of 5.2 g/l (39 mM).     

In vitro distribution of porphyrin metabolites from 10−3 M delta-aminolevulinic acid in primary neural tissue cultures

The distribution of porphyrin metabolites (uro-7-6-5-copro-3-proto-heme) from 10^–3 M delta-aminolevulinic acid (ALA) in neuronal and glial primary cultures was examined by an isotopic technique. In both cell types, 8–9% of the total production (cell + medium) was found in the cells and more than 90% in the medium. In both the cells and the medium the predominant porphyrin fraction was uro + 7 carboxylic. The heme fraction of the cells was also very high. Through the exclusion of ALA-synthetase activity, the Uroporphyria-like-model of hepatic porphyria was demonstrated.     

Production of erythritol and mannitol by Yarrowia lipolytica yeast in media containing glycerol

Glycerol is a by-product generated in large amounts during the production of biofuels. This study presents an alternative means of crude glycerol valorization through the production of erythritol and mannitol. In a shake-flasks experiment in a buffered medium, nine Yarrowia lipolytica strains were examined for polyols production. Three strains (A UV'1, A-15 and Wratislavia K1) were selected as promising producers of erythritol or/and mannitol and used in bioreactor batch cultures and fed-batch mode. Pure and biodiesel-derived crude glycerol media both supplemented (to 2.5 and 3.25?%) and not-supplemented with NaCl were applied. The best results for erythritol biosynthesis were achieved in medium with crude glycerol supplemented with 2.5?% NaCl. Wratislavia K1 strain produced up to 80.0?g?l(-1) erythritol with 0.49?g?g(-1) yield and productivity of 1.0?g?l(-1)?h(-1). Erythritol biosynthesis by A UV'1 and A-15 strains was accompanied by the simultaneous production of mannitol (up to 27.6?g?l(-1)). Extracellular as well as intracellular erythritol and mannitol ratios depended on the glycerol used and the presence of NaCl in the medium. The results from this study indicate that NaCl addition to the medium improves erythritol biosynthesis, and simultaneously inhibits mannitol formation.     

A factorial approach for a sugarcane juice-based low cost culture medium: increasing the astaxanthin production by the red yeast Phaffia rhodozyma

A sugarcane juice-based low cost culture medium was previously explored to produce the carotenoid pigment astaxanthin in liquid culture by the red yeast Phaffia rhodozyma (1300?μg astaxanthin/g of dry yeast and 6500?μg/l whole culture medium). Two peculiar limitations in Phaffia are growth temperature (<26?°C) and lack of sugar osmotolerance. Two advantages are the wide biochemical ability for the assimilation and metabolization of disaccharides and the prompt utilization of simple nitrogen sources. For instance, the sucrolytic/ureolytic enzymatic activities deserves exploration. In order to improve the culture medium composition and the conditions of fermentation for highly oxygenated carotenoids (e.g., astaxanthin) a study was carried out with a factorial design in two steps. As a first step, the production of astaxanthin was studied as a function of the nutrient concentration levels and their interactions. The production increase (μg/l) obtained was 23.0% but at the expense of 16.0% pigment content decrease (μg/g). In the second step, the variables pH and agitation level (OTR, oxygen transfer rate) were optimized and then, both goals were attained: the increase of pigment content (418?μg astaxanthin/g of yeast) as well as the absolute pigment production enhancement (1987?μg/l).     

Biosynthesis of intracellular 5-aminolevulinic acid by a newly identified halotolerant <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Of 23 strains of halotolerant (up to 12% w/v NaCl) photosynthetic bacteria isolated from various sources, one isolate, SH5, accumulated intracellular 5-aminolevulinic acid (ALA) at 0.45 μg/g dry cell wt (DCW) growing aerobically in the dark. The strain was identified as Rhodobacter sphaeroides using 16S rDNA sequencing. Biosynthesis of ALA was enhanced to 14 μg/g DCW using modified glutamate/glucose (50 mM) medium with the addition of 10 mM levulinic acid after 24 h cultivation. Addition of 30 μM Fe²⁺ to this medium increased the yield to 226 μg/g DCW.     

Metabolic engineering of Bacillus subtilis for enhanced production of acetoin

Acetoin is widely used in food and other industries. A bdhA and acoA double-knockout strain of Bacillus subtilis produced acetoin at 0.72?mol/mol, a 16.4?% increased compared to the wild type. Subsequent overexpression of the alsSD operon enhanced the acetolactate synthase activity by 52 and 66?% in growth and stationary phases, respectively. However, deletion of pta gene caused little increase of acetoin production. For acetoin production by the final engineered strain, BSUW06, acetoin productivity was improved from 0.087?g/l?h, using M9 medium plus 30?g glucose/l under micro-aerobic conditions, to 0.273?g/h?l using LB medium plus 50?g glucose/l under aerobic conditions. In fermentor culture, BSUW06 produced acetoin up to 20?g/l.     

An efficient biosurfactant-producing bacterium Selenomonas ruminantium CT2, isolated from mangrove sediment in south of Thailand

Biosurfactant-producing bacteria, isolate CT2, was isolated from mangrove sediment in the south of Thailand. The sequence of the 16S rRNA gene from isolate CT2 showed 100?% similarity with Selenomonas ruminantium. The highest biosurfactant production (5.02?g/l) was obtained when the cells were grown on minimal salt medium containing 15?g/l molasses and 1?g/l commercial monosodium glutamate supplemented with 1?g/l NaCl, 0.1?g/l leucine, 5?% (v/v) inoculum size at 30?°C and 150?rpm after 54?h of cultivation. The biosurfactant obtained by extraction with ethyl acetate showed high surface tension reduction (25.5?mN/m), a small CMC value (8?mg/l), thermal and pH stability with respect to surface tension reduction and emulsification activity and a high level of salt tolerance. The biosurfactant obtained was confirmed as a lipopeptide by using a biochemical test, FT-IR, MNR and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and also had the ability to emulsify oil and enhance PAHs solubility.     

Poly(3-hydroxybutyrate) production from xylose by recombinant Escherichia coli

Several recombinant Escherichia coli strains harboring the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes were used to produce poly(3-hydroxybutyrate), PHB, from xylose. By flask culture of TG1 (pSYL107) in a defined medium containing 20?g/l xylose, PHB concentration of 1.7?g/l was obtained. Supplementation of a small amount of cotton seed hydrolysate or soybean hydrolysate could enhance PHB production by more than two fold. The PHB concentration, PHB content, and PHB yield on xylose obtained by supplementing soybean hydrolysate were 4.4?g/l, 73.9%, and 0.226?g PHB/g xylose, respectively.     

Biosynthesis of δ-aminolevulinic acid from glutamate by Sulfolobus solfataricus

The extremely thermophilic, obligately aerobic bacterium Sulfolobus solfataricus forms the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate by the tRNA-dependent five-carbon pathway. This pathway has been previously shown to occur in plants, algae, and most prokaryotes with the exception of the -group of proteobacteria (purple bacteria). An alternative mode of ALA formation by condensation of glycine and succinyl-CoA occurs in animals, yeasts, fungi, and the -proteobacteria. Sulfolobus and several other thermophilic, sulfur-dependent bacteria, have been variously placed within a subgroup of archaea (archaebacteria) named crenarchaeotes, or have been proposed to comprise a distinct prokaryotic group designated eocytes. On the basis of ribosomal structure and certain other criteria, eocytes have been proposed as predecessors of the nuclear-cytoplasmic descent line of eukaryotes. Because aplastidic eukaryotes differ from most prokaryotes in their mode of ALA formation, and in view of the proposed affiliation of eocytes to eukaryotes, it was of interest to determine how eocytes form ALA. Sulfolobus extracts were able to incorporate label from [1-¹⁴C]glutamate, but not from [2-¹⁴C]glycine, into ALA. Glutamate incorporation was abolished by preincubation of the extract with RNase. Sulfolobus extracts contained glutamate-1-semialdehyde aminotransferase activity, which is indicative of the five-carbon pathway. Growth of Sulfolobus was inhibited by gabaculine, a mechanism-based inhibitor of glutamate-1-semialdehyde aminotransferase, an enzyme of the five-carbon ALA biosynthetic pathway. These results indicate that Sulfolobus uses the five-carbon pathway for ALA formation.Abbreviations AHA 4-amino-5-hexynoic acid - ALA -aminolevulinic acid, Gabaculine, 3-amino-2,3-dihydrobenzoic acid - GSA glutamate 1-semialdehyde     

Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

光合细菌嗜酸柏拉红菌5-氨基乙酰丙酸合成酶基因的克隆与原核表达

5-氨基乙酰丙酸(ALA)可作为除草剂、杀虫剂和植物生长调节剂在农业上应用,但由于其成本较高而限制了它的大面积使用。利用常规基因工程操作方法结合载体介导PCR法(Vecterette PCR)克隆了嗜酸柏拉红菌(Rhodoblastus acidophilus)的5-氨基乙酰丙酸合成酶(ALAS)基因。并将编码ALAS的基因插入到原核表达载体pQE30中,在大肠杆菌不同菌株(E.coli JM109、M15及BL21(DE3))中进行诱导表达。对产物进行SDS-PAGE分析表明,ALAS基因已在细菌中成功表达。使用Ni-NTA亲和层析法对表达的ALAS进行分离、纯化,得到大小约为44kD的ALAS蛋白。通过优化工程菌株的培养条件,建立了发酵生产ALA的方法,其胞外分泌ALA产量达5.379g/L,ALAS酶活力高达333U/min.mg。这是目前国内外利用生物法生产ALA产量最高的报道,为ALA的产业化应用打下了良好的基础。