Production of bioethanol and biodiesel using instant noodle waste

Instant noodle manufacturing waste was used as feedstock to convert it into two products, bioethanol and biodiesel. The raw material was pretreated to separate it into two potential feedstocks, starch residues and palm oil, for conversion to bioethanol and biodiesel, respectively. For the production of bioethanol, starch residues were converted into glucose by α-amylase and glucoamylase. To investigate the saccharification process of the pretreated starch residues, the optimal pretreatment conditions were determined. The bioethanol conversion reached 98.5 % of the theoretical maximum by Saccharomyces cerevisiae K35 fermentation after saccharification under optimized pretreatment conditions. Moreover, palm oil, isolated from the instant noodle waste, was converted into valuable biodiesel by use of immobilized lipase (Novozym 435). The effects of four categories of alcohol, oil-to-methanol ratio, reaction time, lipase concentration and water content on the conversion process were investigated. The maximum biodiesel conversion was 95.4 %.     

Purification and Characterization of Methionine:Glyoxylate Aminotransferase from Brassica carinata and Brassica napus

The first step in the biosynthesis of allylglucosinolate from methionine in Brassica is thought to be the transamination of methionine to 2-keto-4-methylthiobutyrate. By using Q-Sepharose and Red Agarose, followed by high resolution anion exchange chromatography and chromatofocussing, a methionine:glyoxylate aminotransferase (MGAT) was purified to homogeneity from leaves of Brassica carinata var R-4218, and approximately 5000-fold from leaves of Brassica napus var Topas. The final purification was accomplished using nondenaturing polyacrylamide gel electrophoresis. The enzyme has a pl of 4.3, a native molecular mass of 230 to 290 kilodaltons, and a subunit molecular mass of approximately 50 kilodaltons. Four isozymes of the enzyme were identified in the six species of Brassica commonly cultivated. Nonglucosinolate producing species had only low levels of MGAT or an MGAT isozyme which was distinctly different from that in Brassica.     

Plant regeneration from cotyledon protoplasts of Brassica carinata

Protoplasts isolated from cotyledons of Brassica carinata, underwent sustained division when cultured at 5.0 × 10⁴ ml^-1 in modified 8p medium (KM8P) with 1.0% (w/v) Seaplaque agarose. Cell colonies produced callus when agarose droplets, in which the protoplasts were embedded, were transferred to K8 medium with 0.6% (w/v) Sigma Type I or Type VII agarose at day 16, giving a plating efficiency of 1.6%. Seventy percent of the protoplast derived-tissues produced shoot buds after subculture to MS medium containing 3.0% (w/v) sucrose, 1.125 mgl^-1 BAP, 0.035 mgl^-1 GA and 0.6% (w/v) Type I agarose, resulting in shoot formation from 1.1% of the protoplasts originally plated. Protoplast-derived colonies transferred to hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose produced roots. The latter gave rise to shoots after excision from the parent callus and culture on MS medium with 3.0% sucrose, 0.225 mgl^-1 BAP, and 0.6% (w/v) Type I agarose. Shoots regenerated directly from protoplast-derived calli, or indirectly from roots, developed prolific root systems when placed on hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose.Abbreviations BAP 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - K kinetin - NAA -naphthaleneacetic acid - MES 2(N-morpholino)ethanesulphonic acid, 2,iP-6(,-dimethylallyamino) purine - IAA indole-3-acetic acid - Z zeatin - ZR zeatin riboside     

Plant Eegeneration from Protoplasts of Brassica carinata Braun

Protoplasts of Brassica carinata Braun. (accession No. 84A165) were enzymatically isolated from hypocotyls and cotyledons of 3–5 day-old test-tube seedlings or first true leaves taken from greenhouse grown plants at a three-leaf stage. The protoplasts were suspended in a P-B liquid medium solidified with 0.15%–0.3% low melting agarose which formed a thin layer floating on the surface of the liquid medium. The optimum protoplast density was ranging from 5× l03 to 1 × 104/ml. As for the hypocotyl protoplasts, the first division was observed after 48 h in the culture. The division frequency reached 21% and 34% at day 3 and 6 respectively. The initiation of cell division in the case of cotyledon and mesophyll protoplast culture was late, usually at day 5, and the division frequency was also somewhat lower. One week after culture, the cultures were transferred to fluorescent light condition with an intensity of about 1000 lx. A dilution medium DPDK3 was then added and the dilution procedure was repeated at one week interval thereafter. One month after culture, microcalli with 300–500 μm in size were formed. It was also found that in some cases globular embryoid structure protruded on the callus surface. Totally, a 2%–3% plating efficiency was achieved. Shoot regeneration occurred when cotyledon and mesophyll protoplast-derived calli were transferred onto a modified MS medium supplemented with NAA 0.1, BA 3 mg/l. Individual shoots were rooted on a rooting medium supplemented with 0.2 mg/l of IAA. Intact plants with normal morphology were eventually produced.     

Protoplast culture and plant regeneration from Brassica carinata Braun

Protoplasts isolated from hypocotyls of three-day-old seedlings of Brassica carinata (Braun) cv R-2128 were cultured in a modified Nitsch and Nitsch liquid medium containing 13% sucrose, 0.4% Ficoll, 0.25 mg/l BA, 0.5 mg/l NAA and 0.5 mg/l 2,4-D. The density of medium caused the protoplasts and the developing microcalli to float on the surface of the liquid medium whereas all debris and lysed cells sank to the bottom of the culture plate. After 4–6 weeks developing microcalli were approximately 0.5 mm in diameter and were transferred onto MS medium containing 3% sucrose, 0.4% agarose, 200 mg/l casein hydrolysate, 5 mg/l BA and 0.5 mg/l NAA, pH 5.7. Approximately 20% of the calli transferred to this medium produced plantlets.Abbreviations BA 6-Benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashiqe-Skoog     

Synthesis of Brassica carinata from Brassica nigra x Brassica oleracea hybrids obtained by ovary culture

Summary Brassica carinata was synthesized by hybridization between its diploid progenitor species B. nigra and B. oleracea followed by chromosome doubling. Up to 40 times more hybrids were obtained, using ovary culture than with conventional hand pollination. Two hybrids from the cross B. nigra x B. oleracea var alboglabra were raised to maturity. High chromosome pairing was observed in both the hybrids and the amphidiploid. A range of variability for desirable characters is reported in the synthesized amphidiploids.     

Rapid and efficient plant regeneration from hypocotyl protoplasts of Brassica carinata

Protoplasts were isolated from hypocotyls of 7-d-old seedlings of three genotypes of Brassica carinata after enzymatic digestion in cellulase R-10 (0.5%) and pectolyase Y-23 (0.025%). The protoplasts were stabilized with 0.4 M mannitol used as osmoticum, and were cultured in darkness in Kao's liquid medium containing 0.4 M glucose and the growth regulators 2,4-D (1.0 mg/l), NAA (0.1 mg/l) and zeatin riboside (0.5 mg/l). Protoplasts were transferred to 16 h photoperiod conditions after 3 d of dark culture, and the medium was diluted to reduce the osmoticum on the seventh and tenth days of culture. Microcolonies were thus obtained which, upon transfer to MS agarose medium with 2,4-D (0.1 mg/l), BAP (1 mg/l) and 0.1 M sucrose, proliferated further to produce callus clumps. The plating efficiency of the three genotypes varied from 1 to 2%. Calli 2–3 mm in diameter were transferred to MS agarose plates with zeatin (2 mg/l) where they produced shoot buds and shoots with frequencies ranging from 22.5 to 74.2% for the three genotypes. The shoots were rooted in medium with IBA (1 mg/l) and were then established in soil. The time required for protoplast to plant development was 8 to 10 weeks.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -naphthalene acetic acid - BAP 6-Benzylaminopurine - KN Kinetin - 2IP 6-(Gamma, gamma-dimethylallyl-amino)purine     

Production of biodiesel and lactic acid from rapeseed oil using sodium silicate as catalyst

Biodiesel and lactic acid from rapeseed oil was produced using sodium silicate as catalyst. The transesterification in the presence of the catalyst proceeded with a maximum yield of 99.6% under optimized conditions [3% (w/w) sodium silicate, methanol/oil molar ratio 9/1, reaction time 60 min, reaction temperature 60 °C, and stirring rate 250 rpm]. After six consecutive transesterification reactions, the catalyst was collected and used for catalysis of the conversion of glycerol to lactic acid. A maximum yield of 80.5% was achieved when the reaction was carried out at a temperature of 300 °C for 90 min. Thus, sodium silicate is an effective catalyst for transesterification and lactic acid production from the biodiesel by-product, glycerol.     

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa

Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - IBA Indole-3-butyric acid - GA₃ gibberellic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) basal medium     

Histone H3 methylation patterns in Brassica nigra, Brassica juncea, and Brassica carinata species

Core histones are subjected to various post-translational modifications, and one of them, most intensively studied in plants, is the methylation of histone H3. In the majority of analyzed plant species, dimethylation of H3 at lysine 9 (H3K9me2) is detected in heterochromatin domains, whereas methylation of H3 at lysine 4 (H3K4me2) is detected in euchromatin domains. The distribution of H3K9me2 in the interphase nucleus seems to be correlated with genome size, chromatin organization, but also with tissue specificity. In this paper, we present the analysis of the pattern and level of histone H3 methylation for two allotetraploid and one diploid Brassica species. We have found that the pattern of H3K9me2 in interphase nuclei from root meristematic tissue is comparable within the analyzed species and includes both heterochromatin and euchromatin, but the level of modification differs not only among species but even among nuclei in the same phase of the cell cycle within one species. Moreover, the differences in the level of H3K9me2 are not directly coupled with DNA content in the nuclei and are probably tissue specific.     

Synthesis and characterization of poly(3-hydroxyalkanoates) from Brassica carinata oil with high content of erucic acid and from very long chain fatty acids

Pseudomonas aeruginosa produced medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) when grown on substrates containing very long chain fatty acids (VLCFA, C>20). Looking for low cost carbon sources, we tested Brassica carinata oil (erucic acid content 35-48%) as an intact triglyceride containing VLCFA. Oleic (C18:1), erucic (C22:1), and nervonic (C24:1) acids were also employed for mcl-PHA production as model substrates. The polymers obtained were analyzed by GC of methanolyzed samples, GPC, 1H and 13C NMR, ESI MS of partially pyrolyzed samples, and DSC. The repeating units of such polymers were saturated and unsaturated, with a higher content of the latter in the case of the PHA obtained from B. carinata oil. Statistical analysis of the ion intensity in the ESI mass spectra showed that the PHAs from pure fatty acids are random copolymers, while the PHA from B. carinata oil is either a pure polymer or a mixture of polymers. Weight-average molecular weight varied from ca. 56,000 g/mol for the PHA from B. carinata oil and oleic acid, to about 120,000 g/mol for those from erucic and nervonic acids. The PHAs from erucic and nervonic acids were partially crystalline, with rubbery characteristics and a melting point (Tm) of 50°C, while the PHAs from oleic acid and from B. carinata oil afforded totally amorphous materials, with glass transition temperatures (Tg) of -52°C and -47°C, respectively.     

Uptake kinetics of different arsenic species by Brassica carinata

The time- and concentration-dependent uptake kinetics for arsenate and arsenite were determined in 15-day-old excised roots. In both cases, arsenite showed a mono-phasic influx with the isotherm data fitting a linear model better than a non-linear one. The time- and the concentration-dependent uptake of arsenate displayed a hyperbolic kinetic. Greater uptake of arsenate, compared with arsenite, was found especially at lower external substrate concentrations. Competitive inhibition of uptake with phosphate showed that arsenite and arsenate were taken up by different uptake systems because arsenate uptake was strongly inhibited in the presence of phosphate, whereas arsenite uptake was not affected.     

Water-stress and N-nutrition effects on photosynthesis and growth of Brassica carinata

Effects of water and nitrogen (N) supply on growth and photosynthetic response of B. carinata were examined in this study. Plant growth and related characteristics varied significantly in response to the availability of water and nitrogen. B. carinata maximized the utilization efficiency of the most limiting resources by developing physiological adaptations, such as changes in root and leaf development. The utilization of water and N was tightly linked with the availability of each resource. Instantaneous water-use efficiency (WUE) was always greater in plants with high-N nutrition [50, 100, and 150 kg(N) ha^?1] than in the low-N-treated plants [0 kg(N) ha^?1] in all watering treatments. Instantaneous N-use efficiency (PNUE) decreased significantly with increasing water stress in all N treatments. Seed yield is significantly related to PNUE (p>0.05) but not WUE (p<0.05). The positive relationship between leaf net photosynthetic rate (P _N) and seed yield suggests that P _N can be used as an important tool for selection of new strains with high seed yield.     

Production of biodiesel fuel from soybean oil catalyzed by fungus whole-cell biocatalysts in ionic liquids

The methanolysis of soybean oil to produce a fatty acid methyl ester (ME, i.e., biodiesel fuel) was catalyzed by lipase-producing filamentous fungi immobilized on biomass support particles (BSPs) as a whole-cell biocatalyst in the presence of ionic liquids. We used four types of whole-cell biocatalysts: wild-type Rhizopus oryzae producing triacylglycerol lipase (w-ROL), recombinant Aspergillus oryzae expressing Fusarium heterosporum lipase (r-FHL), Candida antarctica lipase B (r-CALB), and mono- and diacylglycerol lipase from A. oryzae (r-mdlB). w-ROL gave the high yield of fatty acid methyl ester (ME) in ionic liquid [Emim][BF₄] or [Bmim][BF₄] biphasic systems following a 24 h reaction. While lipases are known to be severely deactivated by an excess amount of methanol (e.g. 1.5 Mequiv. of methanol against oil) in a conventional system, methanolysis successfully proceeded even with a methanol/oil ratio of 4 in the ionic liquid biphasic system, where the ionic liquids would work as a reservoir of methanol to suppress the enzyme deactivation. When only w-ROL was used as a biocatalyst for methanolysis, unreacted mono-glyceride remained due to the 1,3-positional specificity of R. oryzae lipase. High ME conversion was attained by the combined use of two types of whole-cell biocatalysts, w-ROL and r-mdlB. In a stability test, the activity of w-ROL was reduced to one-third of its original value after incubation in [Bmim][BF₄] for 72 h. The stability of w-ROL in [Bmim][BF₄] was greatly enhanced by cross-linking the biocatalyst with glutaraldehyde. The present study demonstrated that ionic liquids are promising candidates for use as the second solvent in biodiesel fuel production by whole-cell biocatalysts.     

Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata

Shoot organogenesis and plant regeneration were readily achieved from cotyledonary petioles and hypocotyls of Brassica carinata. These explants were used for Agrobacterium-mediated transformation. A construct containing the selectable marker genes, neomycin phosphotransferase II, phosphinothricin acetyl transferase and the reporter gene β-glucuronidase, under the control of a tandem 35S promoter, was used for transformation. Although transformation was achieved with both cotyledonary petioles and hypocotyls, cotyledonary petioles responded best, with 30–50% of the explants producing GUS-positive shoots after selection on 25 mg/l kanamycin. Direct selection on L-phosphinothricin also produced resistant shoots but at a lower frequency (1–2%). Received: 9 April 1997 / Revision received: 3 July 1997 / Accepted: 30 July 1997     

Production of biodiesel from acid waste lard

The objective of the present work was: (i) to enable biodiesel production from acid waste lard; (ii) to study the esterification reaction as possible pre-treatment at different temperatures, catalyst amount and reaction times; (iii) to evaluate biodiesel quality according to EN 14214 after basic transesterification of the pre-treated fat; and (iv) to predict the impact of using such waste as raw material in mixture with soybean oil. Temperature and catalyst amount were the most important reaction conditions which mostly affected biodiesel quality, namely viscosity and purity. The selected pre-treatment conditions were 65 °C, 2.0 wt% H₂SO₄ and 5 h, which allowed obtaining a product with a viscosity of 4.81 mm² s⁻¹ and a purity of 99.6 wt%. The proposed pre-treatment was effective to enable acid wastes as single raw materials for biodiesel production with acceptable quality; however, low yields were obtained (65 wt%). Alkali transesterification of a mixture of waste lard and soybean oil resulted in a product with a purity of 99.8 wt% and a yield of 77.8 wt%, showing that blending might be an interesting alternative to recycle such wastes. Also, because in addition to using conventional and relatively economical processes, some biodiesel properties depending on the raw material composition (such as the iodine value) might even be improved.     

Extraction of oil from microalgae for biodiesel production: A review

The rapid increase of CO(2) concentration in the atmosphere combined with depleted supplies of fossil fuels has led to an increased commercial interest in renewable fuels. Due to their high biomass productivity, rapid lipid accumulation, and ability to survive in saline water, microalgae have been identified as promising feedstocks for industrial-scale production of carbon-neutral biodiesel. This study examines the principles involved in lipid extraction from microalgal cells, a crucial downstream processing step in the production of microalgal biodiesel. We analyze the different technological options currently available for laboratory-scale microalgal lipid extraction, with a primary focus on the prospect of organic solvent and supercritical fluid extraction. The study also provides an assessment of recent breakthroughs in this rapidly developing field and reports on the suitability of microalgal lipid compositions for biodiesel conversion.     

芥菜型油菜与埃塞俄比亚芥杂种基因组原位杂交分析

原产于非洲的埃塞俄比亚芥(Brassica carinata,2n=34,BBCC)具有适应于炎热干旱地区种植等特点,是改良我国芥菜型油菜(B.juncea,2n=36,AABB)的重要种质资源。本研究用基因组原位杂交方法(GISH,Genomic in situ hybridization)分析了芥菜型油菜与埃塞俄比亚芥种间杂种花粉母细胞的染色体分离,发现在后期I染色体主要以17∶18类型分离,其次是16∶19,染色体落后现象偶然发生,其中B染色体组以8∶8的分离比率较高,表明不同来源的B染色体可正常配对分离。本研究表明,芥菜型油菜与埃塞俄比亚芥远缘杂交,通过染色体同源重组(B染色体间),以及部分同源染色体配对交换的方式(A、B、C基因组间),有可能将埃塞俄比亚芥优良遗传成分转移到芥菜型油菜中。