Leafy Biomass Research Articles

Plant Morphoecological Traits, Grass-Weed Interactions and Water Use Efficiencies of Grasses Used for Restoration of African Rangelands

Degradation characterized by depleted vegetation cover is a serious environmental problem in African rangelands. It poses a serious threat to millions of pastoralists and agropastoralists who depend on livestock as a source of livelihood. Consequently, there has been a growing global interest to consolidate efforts to restore degraded ecosystems. For example, the UN decade of Ecosystem Restoration initiative aims at uniting the world behind a common goal of preventing, halting and reversing the degradation of ecosystems. Grass reseeding using native perennial species has been identified as one of the practical ecological strategies for restoring degraded African rangelands, enhancing vegetation cover and forage production. Knowledge of the multifaceted performance of African rangeland grasses in terms of morphoecological traits, interaction with weeds and water use efficiencies is however largely limited and often elusive. Perennial grasses indigenous to African rangelands Cenchrus ciliaris L. (African foxtail grass), Enteropogon macrostachyus (Hochst. Ex A. Rich.) Monro ex Benth. (Bush rye grass) and Eragrostis superba Peyr. (Maasai love grass), were established in an African semi-arid rangeland under natural conditions to fill this knowledge gap. Morphoecological plant traits: aboveground biomass (shoot, leaf and stem) production, plant densities, basal cover, tiller densities and plant height were measured 9 months after establishment. Interaction between the target grass species and weeds and water use efficiencies (WUE) were also determined. Enteropogon macrostachyus displayed significantly higher values for plant densities, tiller densities and basal cover, indices commonly used to estimate the potential of grasses for ecological restoration. Eragrostis superba produced the highest shoot biomass and water use efficiencies. This is attributed to its higher leafy biomass fraction. Higher aboveground biomass production of E. superba demonstrate its suitability for enhancing rangeland productivity. Cenchrus ciliaris suppressed the weeds. This is linked to its aggressive and allelopathic nature. In conclusion, the three perennial grasses displayed distinct morphoecological traits. In order to achieve successful seed-based restoration of degraded African rangelands using native perennial grasses, careful selection species to maximize on their unique traits is recommended. Ultimately, this selection process should match the desired restoration outcomes and subsequent use of the rangeland.

Dynamics in tropical lowland soil nutrients as influenced by leafy biomass of some agroforestry trees

One of the major challenges of the developing countries is the production of sufficient food for the rapidly increasing population, and over cultivation of land resulting to soil infertility. Hence, transfer of nutrients through tree biomass contribute to micro variability in soil fertility and plant growth. A field research was conducted in the forest nursery of the Federal University of Agriculture, Abeokuta (FUNAAB), Nigeria to investigate the response of tropical lowland soil to the application of leafy biomass of some agroforestry tree species; Anogeissus leiocarpus, Enterolobium cyclocarpum, Gliricidia sepium, Leuceana leucocephala and Treculia africana at the rate of 5 tons per hectare (5t ha-1 or 5000 kg ha-1). The soil samples were collected at 2, 4, 6, 8 and 10 weeks after application (WAP). Split plot experimental design was used with the time of soil sampling as main plots, mulch type as sub-plots. Data were statistically analysed using Analysis of Variance (ANOVA) at p =0.05 on pH, organic carbon, total N, P, K, Ca, Mg and Na. L. leucocephala had highest nutrient release values on; total nitrogen (0.019±0.002), calcium (41.07±3.75), sodium (139.0±36.927). Control plots was high in organic carbon (3.12±0.067), as well as pH (6.45±0.029). However, E. cyclocarpus also had highest nutrient contents in potassium (90.0±4.79) and magnesium (152.50±24.575). It is concluded that L. leucocephala had fast decomposition rate and its rapid nutrient release is a good alternative to improving soil nutrients in lowland tropical soil and therefore recommended as a better choice soil improvement agroforestry tree leafy biomass among other species as investigated from the study.

Predicting biogas production from a two-plot fit of extractables and recalcitrants from ligno-cellulosic biomass feedstocks

Anaerobic digestion of lignocellulosic biomass is a sustainable source of energy. Easy to degrade extractive based materials were easy to degrade and produced large volumes of gas reaching 350 to 450 ml/gVS fed. Whereas predominantly lignin bound tropical biomass like leaves produced the less gas. Among cereal straws, finger millet produced maximum gas at 450 ml/gVS while wheat produced 273 ml biogas/gVS. The concept of inflection point has been introduced in this paper where the gas production linearized to a two-plot fit, that is the gas production rate at initial stages, predominantly due to pectic (extractives) inflects to a slower second phase of gas production. It was found that the leafy biomass takes a longer time to degrade to produce 60% of total gas with an inflection point between 45 and 70 days. Pectin based biomass such as peels took 10 to 30 days.

How Cultivar and Extraction Conditions Affect Antioxidants Type and Extractability for Olive Leaves Valorization

Olive leafy biomass constitutes an attractive source of biological substances, such as oleuropein, which can be valuable for the production of functional extracts. The incorporation of this valoriz...

Producing Cyclopropane Fatty Acid in Plant Leafy Biomass via Expression of Bacterial and Plant Cyclopropane Fatty Acid Synthases.

Saturated mid-chain branched fatty acids (SMCBFAs) are widely used in the petrochemical industry for their high oxidative stability and low melting temperature. Dihydrosterculic acid (DHSA) is a cyclopropane fatty acid (CPA) that can be converted to SMCBFA via hydrogenation, and therefore oils rich in DHSA are a potential feedstock for SMCBFA. Recent attempts to produce DHSA in seed oil by recombinant expression of cyclopropane fatty acid synthases (CPFASes) resulted in decreased oil content and poor germination or low DHSA accumulation. Here we explored the potential for plant vegetative tissue to produce DHSA by transiently expressing CPFAS enzymes in leaf. When CPFASes from plant and bacterial origin were transiently expressed in Nicotiana benthamiana leaf, it accumulated up to 1 and 3.7% DHSA in total fatty acid methyl ester (FAME), respectively, which increased up to 4.8 and 11.8%, respectively, when the N. benthamiana endogenous oleoyl desaturase was silenced using RNA interference (RNAi). Bacterial CPFAS expression produced a novel fatty acid with a cyclopropane ring and two carbon-carbon double bonds, which was not seen with plant CPFAS expression. We also observed a small but significant additive effect on DHSA accumulation when both plant and bacterial CPFASes were co-expressed, possibly due to activity upon different oleoyl substrates within the plant cell. Lipidomics analyses found that CPFAS expression increased triacylglycerol (TAG) accumulation relative to controls and that DHSA was distributed across a range of lipid species, including diacylglycerol and galactolipids. DHSA and the novel CPA were present in phosphatidylethanolamine when bacterial CPFAS was expressed in leaf. Finally, when plant diacylglycerol acyltransferase was coexpressed with the CPFASes DHSA accumulated up to 15% in TAG. This study shows that leaves can readily produce and accumulate DHSA in leaf oil. Our findings are discussed in line with current knowledge in leaf oil production for a possible route to DHSA production in vegetative tissue.

Seasonal variations of PM10 — Trace elements, PAHs and Levoglucosan: Rural sugarcane growing area versus coastal urban area in Southeastern Florida, USA. Part II: Elemental concentrations

The largest sugarcane-growing area in the United States is in South Florida. An estimated 7 million tons of dry sugarcane leafy biomass is removed from the fields before harvest by burning the leaves off the sugarcane stalks. Preharvest sugarcane leaf foliage burning is a major source of airborne particulate matter (PM), polycyclic aromatic hydrocarbons (PAHs), biogenic trace elements such as K, volatile hydrocarbons and other pollutants. In one harvesting period, approximately 22 million tons of CO2, a greenhouse gas, is released, 7 million tons from leaf foliage preharvest burning alone and 15 million tons from burning sugarcane bagasse in the sugar mills for power generation. In this study, PM10 was collected from Belle Glade, close to the sugarcane-growing area, and from Delray Beach, an urbanized area along the East Coast of Florida. The PM samples were analyzed for trace elements and close to organic compounds. To elucidate the importance of preharvest sugarcane biomass smoke emissions on air quality, 39 trace elements, 18 PAHs, and levoglucosan were selected to apportion ambient PM constituents. Al, Ca and to a great extend also Mg are the major soil-related trace elements and key markers for fugitive soil dust emissions. Similarly, sea salt aerosol blown from the Atlantic Ocean into Florida added appreciably to PM10 trace elements concentration, especially Na and Cl. Approximately half of the sea salt aerosol (PM10 -portion) by mass is removed from the atmosphere during the transport of ocean air from Delray Beach to Belle Glade. Ambient acid displacement reactions caused a substantial portion of the Cl (˜30%) to be liberated from the sea salt aerosol to the atmosphere during transport as gaseous HCl. The PAH concentrations at Belle Glade were high when the biomass combustion markers levoglucosan and K also showed the highest concentrations. This indicates that during the sugarcane harvest season, when leaf foliage is burned off just before harvest, most of the ambient PAHs associated with PM10 are indeed related to preharvest burning, harvesting machinery emissions, bagasse burning in sugarcane mills, and other related activities that consume biomass and/or fossil fuels for sugarcane harvesting and processing.

Developing Allometric Equations for Estimating Shrub Biomass in a Boreal Fen

Allometric equations for estimating aboveground biomass (AGB) from easily measured plant attributes are unavailable for most species common to mid-continental boreal peatlands, where shrubs comprise a large component of the vegetation community. Our study develops allometric equations for three dominant genera found in boreal fens: Alnus spp. (alder), Salix spp. (willow) and Betula pumila (bog birch). Two different types of local equations were developed: (1) individual equations based on genus/phylogeny, and (2) a general equation that pooled all individuals regardless of genera. The general equation had a R2 = 0.97 (n = 82), and was not significantly different (p > 0.05) than any of the phylogenetic equations. This indicated that a single generalized equation is sufficient in estimating AGB for all three genera occurring in our study area. A closer look at the performance of the general equation revealed that smaller stems were predicted less accurately than larger stems because of the higher variability of leafy biomass found in small individuals. Previously published equations developed in other ecoregions did not perform as well as our local equations.

Yield, Nutritional Composition and Antioxidant Properties of Calocybe indica Cultivated on Wheat Straw Basal Substrate Supplemented with Nitrogenous Tree Leaves

In this study, we cultivated Calocybe indica (C. indica) on wheat straw basal substrate and analysed the influence of supplementation of nitrogenous tree leaves of Syzygium cumini (SCL), Bauhinia variegate (BVL) and Cassia fistula (CFL) on yield, nutritional composition, and antioxidant properties. Wheat straw basal substrate was supplemented with different proportions (25, 50, and 75%) of SCL, BVL and CFL to determine the most effective substrate composition. Results indicated that 25 BVL was the best substrate for producing highest yield (622 g) and biological efficiency (82.93%) of C. indica. Principle component analysis (PCA) was performed to observe the correlation within nutritional parameters (crude fat, crude protein, ash, fibre, carbohydrates, moisture and dry matter) and how these different parameters varied in different treatments used in this study. PC1 and PC2 represented 76.27% of total variation in the observed variables. Supplementation of leafy biomass significantly (p < 0.05) improved the minerals (Na, Mg, K, Ca, Mn, Fe, Zn, Se) and nutritional contents of the C. indica over wheat straw alone. PCA of mineral content (mg/100 g) of the C. indica grown on WS supplemented with nitrogenous tree leaves showed their grouping to PC1 and most of the mineral are positively correlated as depicted in correlation matrix. The impact of different substrates on the antioxidant activities of methanolic extracts of C. indica was also studied. Among the ten treatments, 25 BVL produced the fruiting bodies with high antioxidant properties followed by 25 CFL and 25 SCL. In conclusion, nitrogenous leafy biomass could be an economical, novel and easily accessible nitrogenous supplementation for C. indica cultivation.

Sustainable scenarios for alkaline protein extraction from leafy biomass using green tea residue as a model material

AbstractLeaf protein can be extracted cost‐efficiently using 0.1 mol dm−3 NaOH, but this process is less sustainable due to the generation of large amounts of sodium salts. KOH or Ca(OH)2 are considered as replacements for NaOH, as these salts can be reused. This work evaluates the economic and environmental sustainability of weak alkaline pectin extraction followed by KOH enhanced protein extraction, and Viscozyme® L‐aided pectin extraction followed by Ca(OH)2 enhanced protein extraction. The evaluations are made for green tea residue and are compared to related processes using NaOH. The predicted profits using KOH are comparable to those using NaOH. Environmental sustainability improves for all impact categories in the case of KOH extraction. Further environmental benefits are obtained by substituting conventional K fertilizer with the K‐rich salty waste water from the extraction process. The profits of the process using Ca(OH)2 are highly dependent on the extraction yield of the protein product. Protein extraction yields using Ca(OH)2 need to be higher than 70% to be more profitable than the same process with NaOH. The environmental benefits of Ca(OH)2 extraction include the absence of salty waste water and the net production of heat. This is accompanied by increased electricity consumption. Thus, the impact categories of climate change, fossil and water depletion, and particulate matter formation worsen. Photochemical oxidant formations remain the same, while the other impacts improve. This work has shown the potential and bottlenecks of NaOH, KOH and Ca(OH)2 protein extraction on different types of biomass in terms of environmental and economic sustainability. © 2018 Society of Chemical Industry and John Wiley &amp; Sons, Ltd

Trace element leaching from contaminated willow and poplar biomass – A laboratory study of potential risks

Despite the advantages of phytoextraction, serious risks can be caused by the produced contaminated biomass. Currently, there is a lack of research on the potential risk of storing and handling this biomass. The aim of our study was to verify the losses of potential risk elements (Cd, Cu, Fe, Mn, Pb, Zn) via leaching of short rotation coppice biomass in laboratory batch tests. Harvested woody and leafy biomass was chipped and/or kept compact. The fresh and/or dried biomass was then leached by two types of leachate to simulate rainfalls—neutral water and/or acidified water. Chipped wood was characterised by the highest element release in comparison to whole branches. The leaves were considered more risky from the point of view of element losses. A similar trend of leaching was observed for all elements, except iron. More iron was leached from fresh wood biomass, whilst in the case of the leaves, iron was leached more from dry biomass. Lead was released by 1.4–7.2% from the biomass. Cadmium was leached by 2% from woody biomass, but up to 39% from leaves. The potential losses of these elements during storage and pre-processing should be taken into account in the development of methodologies for the storage and handling of contaminated biomass before its' processing.

Splice Variants of the Castor WRI1 Gene Upregulate Fatty Acid and Oil Biosynthesis When Expressed in Tobacco Leaves.

The plant-specific WRINKLED1 (WRI1) is a member of the AP2/EREBP class of transcription factors that positively regulate oil biosynthesis in plant tissues. Limited information is available for the role of WRI1 in oil biosynthesis in castor bean (Ricinus connunis L.), an important industrial oil crop. Here, we report the identification of two alternatively spliced transcripts of RcWRI1, designated as RcWRI1-A and RcWRI1-B. The open reading frames of RcWRI1-A (1341 bp) and RcWRI1-B (1332 bp) differ by a stretch of 9 bp, such that the predicted RcWRI1-B lacks the three amino acid residues “VYL” that are present in RcWRI1-A. The RcWRI1-A transcript is present in flowers, leaves, pericarps and developing seeds, while the RcWRI1-B mRNA is only detectable in developing seeds. When the two isoforms were individually introduced into an Arabidopsis wri1-1 loss-of-function mutant, total fatty acid content was almost restored to the wild-type level, and the percentage of the wrinkled seeds was largely reduced in the transgenic lines relative to the wri1-1 mutant line. Transient expression of each RcWRI1 splice isoform in N. benthamiana leaves upregulated the expression of the WRI1 target genes, and consequently increased the oil content by 4.3–4.9 fold when compared with the controls, and RcWRI1-B appeared to be more active than RcWRI1-A. Both RcWRI1-A and RcWRI1-B can be used as a key transcriptional regulator to enhance fatty acid and oil biosynthesis in leafy biomass.

Field investigations of selectively treated bamboo species

In the present study, eco-friendly treatment of D. strictus bamboo species was performed using traditional water leaching method and smoking and plant extract (neem oil, cedar oil, jatropha cake extract, aqueous extract of leafy biomass of lantana and jatropha leaves). Control samples were damaged up to 60 % within 3 months and completely destroyed within 6 months of exposure to termites. Results of field investigations were found to be in conformity with laboratory findings of earlier investigations. Though, both traditional treatments of bamboo were able to provide protection better than control sample yet full protection in field was not achieved. Copperised neem oil treated bamboo specimens were found to be the most durable samples up to 3 years of exposure to field conditions.

Temporary Immersion Bioreactors for the Contained Production of Recombinant Proteins in Transplastomic Plants.

Despite the largely maternal inheritance of plastid genomes, the risk of transgene dissemination from transplastomic plants can limit the scope for field cultivation. There is a need for a cost-effective, scalable process to grow large quantities of transplastomic plant biomass for biosynthesis of biopharmaceuticals and other high-value heterologous proteins. Temporary immersion culture is a means of achieving this under fully contained conditions. This method describes the organogenesis of transplastomic Nicotiana tabacum callus in RITA(®) temporary immersion bioreactors to produce rootless leafy biomass, and subsequent total soluble protein extraction, SDS-PAGE, and Western immunoblot analysis of heterologous protein expression. This method can be used for propagation of plastid or nuclear transformants, though is especially suitable for transplastomic biomass, as organogenesis leads to greater expression and accumulation of transplastomic proteins due to increases in chloroplast number and size.

Variability in Fire Prescriptions to Promote Wildlife Foods in the Longleaf Pine Ecosystem

AbstractPrescribed fire is commonly used to restore and maintain the longleaf pine (Pinus palustris Mill.) ecosystem (LLPE). A key function of the LLPE is the provisioning of food for wildlife. Despite the plethora of literature evaluating the effects of fire season and fire-return interval on plant community dynamics, little attention has been given to the response of wildlife foods to fire season or fire-return interval. We measured the availability of key wildlife foods (fleshy fruit [i.e., seed containing a nutritious pericarp] and understory plant biomass) in upland pine forest following dormant-season (December–February) and growing-season (April–June) fires in a chronosequential design. Also, we quantified the relative contributions of the upland hardwood and bottomland hardwood forest types, which often are intentionally suppressed in the LLPE. In 2011 and 2012, we measured understory leafy biomass, biomass of forages selected by whitetailed deer (Odocoileus virginianus Zimm.), and soft mast production chronosequentially in relation to years-since-fire, fire season, and vegetation type in the LLPE at Fort Bragg Military Installation, North Carolina, USA. Understory leafy biomass increased in upland pine and hardwood forests as years-since-fire increased until two years post fire. Selected forages decreased in upland pine forest and increased in upland hardwood forest as time-since-fire increased. In upland pine forests burned during the growing season, 94% of the fruit was detected two years after fire, 6% one year after fire, and 0% the same year as fire. In June, fruit density was greatest in bottomland hardwood forest; in July, fruit density was greatest in dormant-season burned upland pine forest; in August, fruit density was greatest in upland hardwood forest; and in September, fruit density was greatest in upland hardwood and bottomland hardwood forest. Overall summer fruit density (i.e., the sum of fruit density detected each month) was greatest in upland hardwood forest. Understory leafy biomass and deer-selected forages were stable in bottomland hardwood forest because they were not burned, thereby providing a relatively high and stable availability from year to year. Our data demonstrate the importance of diversity in fire season and frequency, and diversity in vegetation types to promote key wildlife foods in the LLPE.

Preparation of gasification feedstock from leafy biomass.

Dried leaves are a potential source of energy although these are not commonly used beside to satisfy daily energy demands in rural areas. This paper aims at preparing a leafy biomass feedstock in the form of briquettes which can be directly used for combustion or to extract the combustible gas using a gasifier. Teak (Tectona grandis) and rubber (Hevea brasiliensis) leaves are considered for the present study. A binder-assisted briquetting technique with tapioca starch as binder is adopted. Properties of these leafy biomass briquettes such as moisture content, calorific value, compressive strength, and shatter index are determined. From the study, briquettes with biomass-to-binder ratio of 3:5 are found to be stable. Higher mass percentage of binder is considered for preparation of briquettes due to the fact that leafy biomasses do not adhere well on densification with lower binder content. Ultimate analysis test is conducted to analyze the gasification potential of the briquettes. Results show that the leafy biomass prepared from teak and rubber leaves has calorific values of 17.5 and 17.8MJ/kg, respectively, which are comparable with those of existing biomass feedstock made of sawdust, rice husk, and rice straw.

Bioethanol production from hemicellulose rich Populus nigra involving recombinant hemicellulases from Clostridium thermocellum

Bioethanol was produced from poplar leafy biomass rich in hemicelluloses content involving recombinant Clostridium thermocellum hemicellulases and pentose sugar utilizing Candida shehatae. FT-IR analysis revealed effective AFEX pretreatment of poplar leaves. Repetitive batch strategy yielded ∼1.5-fold rise in cell biomass and specific activity of both, acetylxylanesterase (Axe) and GH43 hemicellulase. TLC and HPAEC exhibited xylose and arabinose release from hydrolyzed biomass. SSF trial with 1% (wv−1) pretreated poplar and mixed enzymes showed ∼1.5-fold higher ethanol titre as compared with SHF. The shake flask SSF with 5% (wv−1) pretreated poplar furnished 4.56 and 5.43gL−1 ethanol with Axe and mixed enzymes, respectively. Whereas, bioreactor scale-up exhibited ∼1.25-fold increase in ethanol titres (5.68, 6.75gL−1) as compared with shake flask with an yield of 0.295 (gg−1) and 0.351 (gg−1), respectively with Axe and mixed enzymes.

BIOETHANOL PRODUCTION FROM LEAFY BIOMASS OF MANGO (Mangifera indica) INVOLVING NATURALLY ISOLATED AND RECOMBINANT ENZYMES

The present study describes the usage of dried leafy biomass of mango (Mangifera indica) containing 26.3% (w/w) cellulose, 54.4% (w/w) hemicellulose, and 16.9% (w/w) lignin, as a substrate for bioethanol production from Zymomonas mobilis and Candida shehatae. The substrate was subjected to two different pretreatment strategies, namely, wet oxidation and an organosolv process. An ethanol concentration (1.21 g/L) was obtained with Z. mobilis in a shake-flask simultaneous saccharification and fermentation (SSF) trial using 1% (w/v) wet oxidation pretreated mango leaves along with mixed enzymatic consortium of Bacillus subtilis cellulase and recombinant hemicellulase (GH43), whereas C. shehatae gave a slightly higher (8%) ethanol titer of 1.31 g/L. Employing 1% (w/v) organosolv pretreated mango leaves and using Z. mobilis and C. shehatae separately in the SSF, the ethanol titers of 1.33 g/L and 1.52 g/L, respectively, were obtained. The SSF experiments performed with 5% (w/v) organosolv-pretreated substrate along with C. shehatae as fermentative organism gave a significantly enhanced ethanol titer value of 8.11 g/L using the shake flask and 12.33 g/L at the bioreactor level. From the bioreactor, 94.4% (v/v) ethanol was recovered by rotary evaporator with 21% purification efficiency.

Lignocellulosic Fermentation of Wild Grass Employing Recombinant Hydrolytic Enzymes and Fermentative Microbes with Effective Bioethanol Recovery

Simultaneous saccharification and fermentation (SSF) studies of steam exploded and alkali pretreated different leafy biomass were accomplished by recombinant Clostridium thermocellum hydrolytic enzymes and fermentative microbes for bioethanol production. The recombinant C. thermocellum GH5 cellulase and GH43 hemicellulase genes expressed in Escherichia coli cells were grown in repetitive batch mode, with the aim of enhancing the cell biomass production and enzyme activity. In batch mode, the cell biomass (A 600 nm) of E. coli cells and enzyme activities of GH5 cellulase and GH43 hemicellulase were 1.4 and 1.6 with 2.8 and 2.2 U·mg−1, which were augmented to 2.8 and 2.9 with 5.6 and 3.8 U·mg−1 in repetitive batch mode, respectively. Steam exploded wild grass (Achnatherum hymenoides) provided the best ethanol titres as compared to other biomasses. Mixed enzyme (GH5 cellulase, GH43 hemicellulase) mixed culture (Saccharomyces cerevisiae, Candida shehatae) system gave 2-fold higher ethanol titre than single enzyme (GH5 cellulase) single culture (Saccharomyces cerevisiae) system employing 1% (w/v) pretreated substrate. 5% (w/v) substrate gave 11.2 g·L−1 of ethanol at shake flask level which on scaling up to 2 L bioreactor resulted in 23 g·L−1 ethanol. 91.6% (v/v) ethanol was recovered by rotary evaporator with 21.2% purification efficiency.

Production of leafy biomass using temporary immersion bioreactors: an alternative platform to express proteins in transplastomic plants with drastic phenotypes

Chloroplast transformation technology is a promising approach for the production of foreign proteins in plants with expression levels of up to 70 % of total soluble protein (TSP) achieved in tobacco. However, expression of foreign protein in the chloroplast can lead to drastic or even lethal effects in transplastomic plants grown in soil, thereby potentially limiting the applicability of this technology. For instance, previous attempts to express the outer surface protein A (OspA) from Borrelia burgdorferi in tobacco chloroplasts led to plant death when expressed at 10 % TSP. We show here that this earlier transplastomic line, as well as a new plant line, OspA:YFP, expressing OspA fused to the yellow fluorescent protein, can be propagated in temporary immersion bioreactors (TIBs) using AlkaBurst™ technology to produce leafy biomass that expressed OspA at levels of up to 7.6 % TSP, to give a maximum yield of OspA of about 108 mg/L. Our results show that TIBs provide an alternative method for the production of transplastomic biomass expressing proteins toxic for plants and is a particularly useful approach when ‘absolute’ containment is required.

Bioethanol Production Involving Recombinant C. thermocellum Hydrolytic Hemicellulase and Fermentative Microbes

The enhancement of the biomass productivity of Escherichia coli cells harbouring the truncated 903 bp gene designated as glycoside hydrolase family 43 (GH43) from Clostridium thermocellum showing hemicellulase activity along with its further use in simultaneous saccharification and fermentation (SSF) process is described. (Phosphoric acid) H(3)PO(4)-acetone treatment and ammonia fibre expansion (AFEX) were the pretreatment strategies employed on the leafy biomass of mango, poplar, neem and asoka among various substrates owing to their high hemicellulose content. GH43 showed optimal activity at a temperature of 50 °C, pH 5.4 with stability over a pH range of 5.0-6.2. A 4-fold escalation in growth of the recombinant E. coli cells was observed when grown using repeated batch strategy in LB medium supplemented with glucose as co-substrate. Candida shehatae utilizing pentose sugars was employed for bioethanol production. AFEX pretreatment proved to be better over acid-acetone technique. The maximum ethanol concentration (1.44 g/L) was achieved for AFEX pretreated mango (1%, w/v) followed by poplar with an ethanol titre (1.32 g/L) in shake flask experiments. A 1.5-fold increase in ethanol titre (2.11 g/L) was achieved with mango (1%, w/v) in a SSF process using a table top 2-L bioreactor with 1 L working volume.