Electrodialytic separation of levulinic acid catalytically synthesized from woody biomass for use in microbial conversion

Levulinic acid (LA) is produced by the catalytic conversion of a variety of woody biomass. To investigate the potential use of desalting electrodialysis (ED) for LA purification, electrodialytic separation of levulinate from both reagent and cedar‐derived LA solution (40–160 g L^?1) was demonstrated. When using reagent LA solution with pH5.0–6.0, the recovery rates of levulinate ranged from 68 to 99%, and the energy consumption for recovery of 1 kg of levulinate ranged from 0.18 to 0.27 kWh kg^?1. With cedar‐derived LA solution (pH6.0), good agreement in levulinate recovery (88–99%), and energy consumption (0.18–0.22 kWh kg^?1) were observed in comparison to the reagent LA solutions, although a longer operation time was required due to some impurities. The application of desalting ED was favorable for promoting microbial utilization of cedar‐derived LA. From 0.5 mol L^?1 of the ED‐concentrated sodium levulinate solution, 95.6% of levulinate was recovered as LA calcium salt dihydrate by crystallization. This is the first report on ED application for LA recovery using more than 20 g L^?1 LA solutions (40–160 g L^?1). © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:448–453, 2017     

合成微生物群落用于木质纤维素生物质转化的研究进展

木质纤维素在自然界中的储量可观,是生物燃料生产的重要来源。联合生物加工(consolidated bioprocessing)指在不添加酶的情况下,将木质纤维素“一步”转化为生物燃料的过程,在能源危机日益严重的今天具有重要的应用价值。合成微生物群落(synthetic microbial consortia)是由两种或多种纯培养微生物(野生菌株或工程菌株)共同培养而形成的菌群,具有复杂性低、稳定性高等优点,通过协调微生物之间的相互作用以及整个生态系统的稳定,从而实现特定的功能。近年来,合成生物学的快速发展有利于开发新的方法和工具用于合成微生物群落的构建及优化,促进其在联合生物加工方面的应用。本文聚焦于木质纤维素的联合生物加工,综述了合成微生物群落在该领域的研究进展。简单介绍了系统生物学为合成微生物群落的设计提供指导,详细介绍了合成微生物群落的设计原则、优化工具和在实际生产中的应用与挑战,为木质纤维素的联合生物加工提供借鉴意义。     

Intensive use of biomass feedstock in ethanol conversion: The alcohol-water, vapor-phase separation

Fermentation of ethanol in a system whereby the biomass is used intensively (both to separate alcohol from water by vapor phase adsorption and to serve as the feedstock) is shown to be possible on theoretical grounds when the biomass is grain. The rationale for a vapor-phase adsorption process as an alternative to distillation is shown to be energetically valid above 84 wt % ethanol. The capacity of grain in new vapor-phase ambient adsorption processes was estimated experimentally with the finding that sufficient capacity exists for the intensive use but that the adsorption is dynamically controlled so that the grain form and particle size are important. Pretreatments such as explosive dehydration improve the transfer of water to the grain in adsorption with potential improvement in the efficiency of liquefaction and saccharification. At room temperature, these sorbents are not perfectly selective for water but adsorb ethanol which will be carried to the liquefaction, saccharification, and fermentation with the feedstock.     

A simple pentose assay for biomass conversion studies

Summary A colorimetric method was modified for monitoring pentose release and utilization in the hydrolysis and fermentation of biomass substrates to fuels and chemicals. The proposed assay was specific for pentose monomers. Quantitation of pentoses by the assay method was not significantly interfered by other lignocellulosic components, common fermentation medium ingredients, and major volatile fermentation products encountered in biomass conversion processes. The assay procedure did not require sample pretreatment (e.g. deproteinization, desalting, or furfural extraction). Sugar estimation basing on the present assay correlated well with conventional sugar analysis by high performance liquid chromatography.     

A review of biomass quality research relevant to the use of poplar and willow for energy conversion

It has been recognized that the chemical and physical properties of biomass feedstocks can play an important role in the efficiency of most energy conversion processes. These properties include the ratio of bark to wood, moisture content, specific gravity, calorific or heating value, and the relative content of extractives, α-cellulose, hemicellulose, and lignin.A review of the literature dealing with the quality of poplar and willow biomass feedstock for energy conversion revealed that considerable variation existed in many of these traits. This variation may make it possible to improve the quality of feedstock through breeding and selection.Little information exists with respect to heritability (both in the broad sense and narrow sense), the genetic correlation between characters, and the presence of genotype-environment interaction. A better understanding of these parameters is essential if the apparent variability is to be used to improve biomass quality.     

Development of water requirement factors for biomass conversion pathway

Published data were used to develop an integrated spreadsheet-based model to estimate total water requirement for 12 biomass conversion pathways. The water requirement for crop production was attributed only to the grains in the estimates since agricultural residues are produced irrespective of their use for fuel or electricity. Corn stover- and wheat straw-based ethanol production pathways are water efficient, requiring only 0.3 l, whereas biopower production pathways (i.e. direct combustion and bio-oil production) require about 0.8-0.9 l of water per MJ. Wheat- and corn-based ethanol production pathways consume 77 and 108 l of water per MJ, respectively. Utilization of switchgrass for production of ethanol, biopower through the direct combustion, and pyrolysis consume 128, 187 and 229 l of water per MJ, respectively. Biodiesel production from canola seed consumes 124 l of water per MJ. Corn stover- and wheat straw-based conversion pathways are most water efficient.     

Advancements and future directions in enzyme technology for biomass conversion

Enzymatic hydrolysis of pre-treated lignocellulosic biomass is an ideal alternative to acid hydrolysis for bio-ethanol production, limited primarily by pre-treatment requirements and economic considerations arising from enzyme production costs and specific activities. The quest for cheaper and better enzymes has prompted years of bio-prospecting, strain optimization through genetic engineering, enzyme characterization for simple and complex lignocellulosic feedstock, and the development of pre-treatment strategies to mitigate inhibitory effects. The recent shift to systematic characterizations of de novo mixtures of purified proteins is a promising indicator of maturation within this field of study, facilitating progression towards feedstock assay-based rapid enzyme mixture optimization. It is imperative that international standards be developed to enable meaningful comparisons between these studies and the construction of a database of enzymatic activities and kinetics, aspects of which are explored here-in. Complementary efforts to improve the economic viability of enzymatic hydrolysis through process integration and reactor design are also considered, where membrane-confinement shows significant promise despite the associated technological challenges. Significant advancements in enzyme technology towards the economic conversion of lignocellulosic biomass should be expected within the next few years as systematic research in enzyme activities conforms to that of traditional reaction engineering.     

The diversity of glycosylation of cellobiohydrolase I from Trichoderma reesei determined with mass spectrometry

Cellulases are glycosylated enzymes that have wide applications in fields like biofuels. It has been widely accepted that glycosylation of cellulases impact their performance. Trichoderma reesei is the most important cellulase-producer and cellobiohydrolase I (CBHI) is the most important cellulase from T. reesei. Therefore, the glycosylation of T. reesei CBHI has been a focus of research. However, investigations have been focused on N-glycosylation of three of the four potential glycosylation sites, as well as O-glycosylation on the linker region, while a full picture of glycosylation of T. reesei CBHI is still needed. In this work, with extensive mass spectrometric investigations on CBHI from two T. reesei strains grown under three conditions, several new discoveries were made: 1) N45 and N64 are N-glycosylated with high mannose type glycans; 2) the catalytic domain of CBHI is extensively O-glycosylated with hexoses and N-acetylhexosamines; 3) experimental evidence on the mannosylation of carbohydrate binding domain (other than the linker adjacent region) was found. With structural analysis, we found several glycosylation sites (such as T383, S8, and S46) are located at the openings of the substrate-binding tunnel, and potentially involve in the binding of cellulose. These investigations provide a full and comprehensive picture on the glycosylation of CBHI from T. reesei, which benefits the engineering of CBHI by raising potential sites for modification.     

Appropriate glycosylation of recombinant proteins for human use

One of the commonest and least well understood posttranslational modifications of proteins is their glycosylation. Human glycoproteins are glycosylated with a bewilderingly heterogeneous array of complex N- and O-linked glycans, which are the product of the coordinated activity of enzymes resident in the endoplasmic reticulum and Golgi apparatus of the cell. Glycosylation of proteins is highly regulated and changes during differentiation, development, under different physiological—and cell culture—conditions and in disease. The glycosylation of recombinant proteins, especially those destined for potential administration to human subjects, is of critical importance. Glycosylation profoundly affects biological activity, function, clearance from circulation, and crucially, antigenicity. The cells of nonhuman species do not glycosylate their proteins in the same way as human cells do. In many cases, the differences are profound. Overall, the species most distant to humans in evolutionary terms, such as bacteria, yeasts, fungi, insects and plants—the species used most commonly in expression systems—have glycosylation repertoires least like our own. This review gives a brief overview of human N- and O-linked protein glycosylation, summarizes what is known of the glycosylation potential of the cells of nonhuman species, and presents the implications for the biotechnology industry.     

Identification of glycan structure and glycosylation sites in cellobiohydrolase II and endoglucanases I and II from Trichoderma reesei

Mass spectrometric techniques combined with enzymatic digestions were applied to determine the glycosylation profiles of cellobiohydrolase (CBH II) and endoglucanases (EG I, II) purified from filamentous fungus Trichoderma reesei. Electrospray mass spectrometry (ESMS) analyses of the intact cellulases revealed the microheterogeneity in glycosylation where glycoforms were spaced by hexose units. These analyses indicated that glycosylation accounted for 12-24% of the molecular mass and that microheterogeneity in both N- and O-linked glycans was observed for each glycoprotein. The identification of N-linked attachment sites was carried out by MALDI-TOF and capillary liquid chromatography-ESMS analyses of tryptic digests from each purified cellulase component with and without PNGase F incubation. Potential tryptic glycopeptide candidates were first detected by stepped orifice-voltage scanning and the glycan structure and attachment site were confirmed by tandem mass spectrometry. For purified CBH II, 74% of glycans found on Asn310 were high mannose, predominantly Hex(7-9)GlcNAc(2), whereas the remaining amount was single GlcNAc; Asn289 had 18% single GlcNAc occupancy, and Asn14 remained unoccupied. EG I presented N-linked glycans at two out of the six potential sites. The Asn56 contained a single GlcNAc residue, and Asn182 showed primarily a high-mannose glycan Hex(8)GlcNAc(2) with only 8% being occupied with a single GlcNAc. Finally, EG II presented a single GlcNAc residue at Asn103. It is noteworthy that the presence of a single GlcNAc in all cellulase enzymes investigated and the variability in site occupancy suggest the secretion of an endogenous endo H enzyme in cultures of T. reesei.     

Implications of land‐use change to Short Rotation Forestry in Great Britain for soil and biomass carbon

Land‐use change can have significant impacts on soil and aboveground carbon (C) stocks and there is a clear need to identify sustainable land uses which maximize C mitigation potential. Land‐use transitions from agricultural to bioenergy crops are increasingly common in Europe with one option being Short Rotation Forestry (SRF). Research on the impact on C stocks of the establishment of SRF is limited, but given the potential for this bioenergy crop in temperate climates, there is an evident knowledge gap. Here, we examine changes in soil C stock following the establishment of SRF using combined short (30 cm depth) and deep (1 m depth) soil cores at 11 sites representing 29 transitions from agriculture to SRF. We compare the effects of tree species including 9 coniferous, 16 broadleaved and 4 Eucalyptus transitions. SRF aboveground and root biomass were also estimated in 15 of the transitions using tree mensuration data allowing assessments of changes in total ecosystem C stock. Planting coniferous SRF, compared to broadleaved and Eucalyptus SRF, resulted in greater accumulation of litter and overall increased soil C stock relative to agricultural controls. Though broadleaved SRF had no overall effect on soil C stock, it showed the most variable response suggesting species‐specific effects and interactions with soil types. While Eucalyptus transitions induced a reduction in soil C stocks, this was not significant unless considered on a soil mass basis. Given the relatively young age and limited number of Eucalyptus plantations, it is not possible to say whether this reduction will persist in older stands. Combining estimates of C stocks from different ecosystem components (e.g., soil, aboveground biomass) reinforced the accumulation of C under coniferous SRF, and indicates generally positive effects of SRF on whole‐ecosystem C. These results fill an important knowledge gap and provide data for modelling of future scenarios of LUC.     

Extremely thermophilic microorganisms for biomass conversion: status and prospects

Many microorganisms that grow at elevated temperatures are able to utilize a variety of carbohydrates pertinent to the conversion of lignocellulosic biomass to bioenergy. The range of substrates utilized depends on growth temperature optimum and biotope. Hyperthermophilic marine archaea (T(opt)>or=80 degrees C) utilize alpha- and beta-linked glucans, such as starch, barley glucan, laminarin, and chitin, while hyperthermophilic marine bacteria (T(opt)>or=80 degrees C) utilize the same glucans as well as hemicellulose, such as xylans and mannans. However, none of these organisms are able to efficiently utilize crystalline cellulose. Among the thermophiles, this ability is limited to a few terrestrial bacteria with upper temperature limits for growth near 75 degrees C. Deconstruction of crystalline cellulose by these extreme thermophiles is achieved by 'free' primary cellulases, which are distinct from those typically associated with large multi-enzyme complexes known as cellulosomes. These primary cellulases also differ from the endoglucanases (referred to here as 'secondary cellulases') reported from marine hyperthermophiles that show only weak activity toward cellulose. Many extremely thermophilic enzymes implicated in the deconstruction of lignocellulose can be identified in genome sequences, and many more promising biocatalysts probably remain annotated as 'hypothetical proteins'. Characterization of these enzymes will require intensive effort but is likely to generate new opportunities for the use of renewable resources as biofuels.     

Reactor scale up for biological conversion of cellulosic biomass to ethanol

The absence of a systematic scale-up approach for biological conversion of cellulosic biomass to commodity products is a significant bottleneck to realizing the potential benefits offered by such conversion. Motivated by this, we undertook to develop a scale-up approach for conversion of waste paper sludge to ethanol. Physical properties of the system were measured and correlations were developed for their dependence upon cellulose conversion. Just-suspension of solid particles was identified as the scale up criterion based on experiments at lab scale. The impeller speed for just solids suspension at large scale was predicted using computational fluid dynamics simulations. The scale-up strategy was validated by analyzing mixing requirements such as solid–liquid mass transfer under the predicted level of agitation at large scale. The scale-up approach enhances the prediction of reactor performance and helps provide guidelines for the analysis and design of large scale bioreactors based on bench scale experimentation.     

Protons @ interfaces: Implications for biological energy conversion

The review focuses on the anisotropy of proton transfer at the surface of biological membranes. We consider (i) the data from “pulsed” experiments, where light-triggered enzymes capture or eject protons at the membrane surface, (ii) the electrostatic properties of water at charged interfaces, and (iii) the specific structural attributes of proton-translocating enzymes. The pulsed experiments revealed that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with the increase in their electric charge, it was concluded that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. The barrier could arise owing to the water polarization at the negatively charged membrane surface. The barrier height depends linearly on the charge of penetrating ions; for protons, it has been estimated as about 0.12 eV. While the proton exchange between the surface and the bulk aqueous phase is retarded by the interfacial barrier, the proton diffusion along the membrane, between neighboring enzymes, takes only microseconds. The proton spreading over the membrane is facilitated by the hydrogen-bonded networks at the surface. The membrane-buried layers of these networks can eventually serve as a storage/buffer for protons (proton sponges). As the proton equilibration between the surface and the bulk aqueous phase is slower than the lateral proton diffusion between the “sources” and “sinks”, the proton activity at the membrane surface, as sensed by the energy transducing enzymes at steady state, might deviate from that measured in the adjoining water phase. This trait should increase the driving force for ATP synthesis, especially in the case of alkaliphilic bacteria.     

Biomass estimation models,biomass storage and ecosystem carbon stock in sweet orange orchards: Implications for land use management

Sweet orange has great socioeconomic value in India and other parts of the world for their important role in human diet and other properties like sweet flavour, sweet aroma, source of vitamin C etc. Despite its numerous commercial values, and large acreages under cultivation little has been studied on the role of sweet orange orchards in carbon management and environmental sustainability. Therefore, the present study was conducted to (1) develop appropriate models for estimation of sweet orange tree biomass, and (2) assess biomass and ecosystem carbon stock for sweet orange orchards in North East India. Allometric models for biomass estimation were developed using data from 58 harvested orange trees. The height-diameter relationships and allometric scaling between above-ground biomass (AGB), culm height (H) and diameter at breast height (D) were examined using various models. Total biomass carbon and soil organic carbon stock of the sweet orange orchard were estimated at 7.69 and 100.2 Mg C ha^?1 respectively. Our finding on biomass carbon stock of the sweet orange orchard was comparable with other fruit orchards across the world. However, the age of the orchard and management systems are two major determinants for carbon sink potential of such systems. We recommend upscaling of sweet orange based agroforestry for restoration of degraded shifting cultivated lands in North East India for environmental sustainability and socioeconomic upliftment of the farmers.     

A review of thermal-chemical conversion of lignocellulosic biomass in China

Biomass, a renewable, sustainable and carbon dioxide neutral resource, has received widespread attention in the energy market as an alternative to fossil fuels. Thermal-chemical conversion of biomass to produce biofuels is a promising technology with many commercial applications. This paper reviewed the state-of-the-art research and development of thermal-chemical conversion of biomass in China with a special focus on gasification, pyrolysis, and catalytic transformation technologies. The advantages and disadvantages, potential of future applications, and challenges related to these technologies are discussed. Conclusively, these transformation technologies for the second-generation biofuels with using non-edible lignocellulosic biomass as feedstocks show prosperous perspective for commercial applications in near future.