Biotechnology for Biofuels

Fosses, Aurélie; Maté, Maria; Franche, Nathalie; Liu, Nian; Denis, Yann; Borne, Romain; de Philip, Pascale; Fierobe, Henri-Pierre; Perret, Stéphanie

doi: 10.1186/s13068-017-0933-7pmid: 29093754

Abstract Background Like a number of anaerobic and cellulolytic Gram-positive bacteria, the model microorganism Ruminiclostridium cellulolyticum produces extracellular multi-enzymatic complexes called cellulosomes, which efficiently degrade the crystalline cellulose. Action of the complexes on cellulose releases cellobiose and longer cellodextrins but to date, little is known about the transport and utilization of the produced cellodextrins in the bacterium. A better understanding of the uptake systems and fermentation of sugars derived from cellulose could have a major impact in the field of biofuels production. Results We characterized a putative ABC transporter devoted to cellodextrins uptake, and a cellobiose phosphorylase (CbpA) in R. cellulolyticum. The genes encoding the components of the ABC transporter (a binding protein CuaA and two integral membrane proteins) and CbpA are expressed as a polycistronic transcriptional unit induced in the presence of cellobiose. Upstream, another polycistronic transcriptional unit encodes a two-component system (sensor and regulator), and a second binding protein CuaD, and is constitutively expressed. The products might form a three-component system inducing the expression of cuaABC and cbpA since we showed that CuaR is able to recognize the region upstream of cuaA. Biochemical analysis showed that CbpA is a strict cellobiose phosphorylase inactive on longer cellodextrins; CuaA binds to all cellodextrins (G2–G5) tested, whereas CuaD is specific to cellobiose and presents a higher affinity to this sugar. This results are in agreement with their function in transport and signalization, respectively. Characterization of a cuaD mutant, and its derivatives, indicated that the ABC transporter and CbpA are essential for growth on cellobiose and cellulose. Conclusions For the first time in a Gram-positive strain, we identified a three-component system and a conjugated ABC transporter/cellobiose phosphorylase system which was shown to be essential for the growth of the model cellulolytic bacterium R. cellulolyticum on cellobiose and cellulose. This efficient and energy-saving system of transport and phosphorolysis appears to be the major cellobiose utilization pathway in R. cellulolyticum, and seems well adapted to cellulolytic life-style strain. It represents a new way to enable engineered strains to utilize cellodextrins for the production of biofuels or chemicals of interest from cellulose.

Ku, Jason T.;Simanjuntak, Wiwik;Lan, Ethan I.

doi: 10.1186/s13068-017-0978-7pmid: 29213330

Abstract Background n-Butyraldehyde is a high-production volume chemical produced exclusively from hydroformylation of propylene. It is a versatile chemical used in the synthesis of diverse C4–C8 alcohols, carboxylic acids, esters, and amines. Its high demand and broad applications make it an ideal chemical to be produced from biomass. Results An Escherichia coli strain was engineered to produce n-butyraldehyde directly from glucose by expressing a modified Clostridium CoA-dependent n-butanol production pathway with mono-functional Coenzyme A-acylating aldehyde dehydrogenase (Aldh) instead of the natural bifunctional aldehyde/alcohol dehydrogenase. Aldh from Clostridium beijerinckii outperformed the other tested homologues. However, the presence of native alcohol dehydrogenase led to spontaneous conversion of n-butyraldehyde to n-butanol. This problem was addressed by knocking out native E. coli alcohol dehydrogenases, significantly improving the butyraldehyde-to-butanol ratio. This ratio was further increased reducing media complexity from Terrific broth to M9 media containing 2% yeast extract. To increase production titer, in situ liquid–liquid extraction using dodecane and oleyl alcohol was investigated. Results showed oleyl alcohol as a better extractant, increasing the titer of n-butyraldehyde produced to 630 mg/L. Conclusion This study demonstrated n-butyraldehyde production from glucose. Through sequential strain and condition optimizations, butyraldehyde-to-butanol ratio was improved significantly compared to the parent strain. Results from this work may serve as a basis for further development of renewable n-butyraldehyde production.

Ho, Ping-Wei; Swinnen, Steve; Duitama, Jorge; Nevoigt, Elke

doi: 10.1186/s13068-016-0696-6pmid: 28053667

Abstract Background Glycerol is an abundant by-product of biodiesel production and has several advantages as a substrate in biotechnological applications. Unfortunately, the popular production host Saccharomyces cerevisiae can barely metabolize glycerol by nature. Results In this study, two evolved derivatives of the strain CEN.PK113-1A were created that were able to grow in synthetic glycerol medium (strains PW-1 and PW-2). Their growth performances on glycerol were compared with that of the previously published evolved CEN.PK113-7D derivative JL1. As JL1 showed a higher maximum specific growth rate on glycerol (0.164 h−1 compared to 0.119 h−1 for PW-1 and 0.127 h−1 for PW-2), its genomic DNA was subjected to whole-genome resequencing. Two point mutations in the coding sequences of the genes UBR2 and GUT1 were identified to be crucial for growth in synthetic glycerol medium and subsequently verified by reverse engineering of the wild-type strain CEN.PK113-7D. The growth rate of the resulting reverse-engineered strain was 0.130 h−1. Sanger sequencing of the GUT1 and UBR2 alleles of the above-mentioned evolved strains PW-1 and PW-2 also revealed one single-point mutation in these two genes, and both mutations were demonstrated to be also crucial and sufficient for obtaining a maximum specific growth rate on glycerol of ~0.120 h−1. Conclusions The current work confirmed the importance of UBR2 and GUT1 as targets for establishing glycerol utilization in strains of the CEN.PK family. In addition, it shows that a growth rate on glycerol of 0.130 h−1 can be established in reverse-engineered CEN.PK strains by solely replacing a single amino acid in the coding sequences of both Ubr2 and Gut1.

Suckling, Ian D.; Jack, Michael W.; Lloyd, John A.; Murton, Karl D.; Newman, Roger H.; Stuthridge, Trevor R.; Torr, Kirk M.; Vaidya, Alankar A.

doi: 10.1186/s13068-017-0748-6pmid: 28293291

Abstract Background Conversion of softwoods into sustainable fuels and chemicals is important for parts of the world where softwoods are the dominant forest species. While they have high theoretical sugar yields, softwoods are amongst the most recalcitrant feedstocks for enzymatic processes, typically requiring both more severe pretreatment conditions and higher enzyme doses than needed for other lignocellulosic feedstocks. Although a number of processes have been proposed for converting softwoods into sugars suitable for fuel and chemical production, there is still a need for a high-yielding, industrially scalable and cost-effective conversion route. Results We summarise work leading to the development of an efficient process for the enzymatic conversion of radiata pine (Pinus radiata) into wood sugars. The process involves initial pressurised steaming of wood chips under relatively mild conditions (173 °C for 3–72 min) without added acid catalyst. The steamed chips then pass through a compression screw to squeeze out a pressate rich in solubilised hemicelluloses. The pressed chips are disc-refined and wet ball-milled to produce a substrate which is rapidly saccharified using commercially available enzyme cocktails. Adding 0.1% polyethylene glycol during saccharification was found to be particularly effective with these substrates, reducing enzyme usage to acceptable levels, e.g. 5 FPU/g OD substrate. The pressate is separately hydrolysed using acid, providing additional hemicellulose-derived sugars, for an overall sugar yield of 535 kg/ODT chips (76% of theoretical). The total pretreatment energy input is comparable to other processes, with the additional energy for attrition being balanced by a lower thermal energy requirement. This pretreatment strategy produces substrates with low levels of fermentation inhibitors, so the glucose-rich mainline and pressate syrups can be fermented to ethanol without detoxification. The lignin from the process remains comparatively unmodified, as evident from the level of retained β-ether interunit linkages, providing an opportunity for conversion into saleable co-products. Conclusions This process is an efficient route for the enzymatic conversion of radiata pine, and potentially other softwoods, into a sugar syrup suitable for conversion into fuels and chemicals. Furthermore, the process uses standard equipment that is largely proven at commercial scale, de-risking process scale-up.

Gao, Ruiling; Li, Zifu; Zhou, Xiaoqin; Cheng, Shikun; Zheng, Lei

doi: 10.1186/s13068-017-0942-6pmid: 29093751

Abstract Background The sustainability of microbial lipids production from traditional carbon sources, such as glucose or glycerol, is problematic given the high price of raw materials. Considerable efforts have been directed to minimize the cost and find new alternative carbon sources. Volatile fatty acids (VFAs) are especially attractive raw materials, because they can be produced from a variety of organic wastes fermentation. Therefore, the use of volatile fatty acids as carbon sources seems to be a feasible strategy for cost-effective microbial lipid production. Results Lipid accumulation in Y. lipolytica using synthetic and food waste-derived VFAs as substrates was systematically compared and evaluated in batch cultures. The highest lipid content obtained with acetic, butyric, and propionic acids reached 31.62 ± 0.91, 28.36 ± 0.74, and 28.91 ± 0.66%, respectively. High concentrations of VFA inhibited cell growth in the following order: butyric acid > propionic acid > acetic acid. Within a 30-day experimental period, Y. lipolytica could adapt up to 20 g/L acetic acid, whereas the corresponding concentration of propionic acid and butyric acid were 10 and 5 g/L, respectively. Cultures on a VFA mixture showed that the utilization of different types of VFA by Y. lipolytica was not synchronized but rather performed in a step-wise manner. Although yeast fermentation is an exothermic process, and the addition of VFA will directly affect the pH of the system by increasing environmental acidity, cultures at a cultivation temperature of 38 °C and uncontrolled pH demonstrated that Y. lipolytica had high tolerance in the high temperature and acidic environment when a low concentration (2.5 g/L) of either synthetic or food waste-derived VFA was used. However, batch cultures fed with food fermentate yielded lower lipid content (18.23 ± 1.12%) and lipid productivity (0.12 ± 0.02 g/L/day). The lipid composition obtained with synthetic and food waste-derived VFA was similar to commercial biodiesel feedstock. Conclusions This work demonstrated the feasibility of utilizing synthetic and food waste-derived VFA for lipid production by Y. lipolytica. The good adaptability of Y. lipolytica to the high temperature and acidic environment further illustrated its considerable potential for practical application.

Yuan, Pengyi; Kim, Younggy

doi: 10.1186/s13068-017-0754-8pmid: 28331546

Abstract Background Microbial electrolysis cells (MECs) use bioelectrochemical reactions to remove organic contaminants at the bioanode and produce hydrogen gas at the cathode. High local pH conditions near the cathode can also be utilized to produce struvite from nutrient-rich wastewater. This beneficial aspect was investigated using lab-scale MECs fed with dewatering centrate collected at a local wastewater treatment plant. The main objective was to improve phosphorus recovery by examining various cathode configurations and electric current conditions. Results The stainless steel mesh (SSM) cathode was relatively inefficient to achieve complete phosphorus recovery because struvite crystals were smaller (a few to tens of micrometers) than the open space between mesh wires (80 µm). As a result, the use of multiple pieces of SSM also showed a limited improvement in the phosphorus recovery up to only 68% with 5 SSM pieces. Readily available organic substrates were not sufficient in the dewatering centrate, resulting in relatively low electric current density (mostly below 0.2 A/m2). The slow electrode reaction did not provide sufficiently high pH conditions near the cathode for complete recovery of phosphorus as struvite. Based on these findings, additional experiments were conducted using stainless steel foil (SSF) as the cathode and acetate (12 mM) as an additional organic substrate for exoelectrogens at the bioanode. With the high electric current (>2 A/m2), a thick layer of struvite crystals was formed on the SSF cathode. The phosphorus recovery increased to 96% with the increasing MEC operation time from 1 to 7 days. With the high phosphorus recovery, estimated energy requirement was relatively low at 13.8 kWh (with acetate) and 0.30 kWh (without acetate) to produce 1 kg struvite from dewatering centrate. Conclusions For efficient phosphorus recovery from real wastewater, a foil-type cathode is recommended to avoid potential losses of small struvite crystals. Also, presence of readily available organic substrates is important to maintain high electric current and establish high local pH conditions near the cathode. Struvite precipitation was relatively slow, requiring 7 days for nearly complete removal (92%) and recovery (96%). Future studies need to focus on shortening the time requirement.

Li, Wan-Chen; Huang, Chien-Hao; Chen, Chia-Ling; Chuang, Yu-Chien; Tung, Shu-Yun; Wang, Ting-Fang

doi: 10.1186/s13068-017-0825-xpmid: 28690679

Abstract Background Trichoderma reesei (Ascomycota, Pezizomycotina) QM6a is a model fungus for a broad spectrum of physiological phenomena, including plant cell wall degradation, industrial production of enzymes, light responses, conidiation, sexual development, polyketide biosynthesis, and plant–fungal interactions. The genomes of QM6a and its high enzyme-producing mutants have been sequenced by second-generation-sequencing methods and are publicly available from the Joint Genome Institute. While these genome sequences have offered useful information for genomic and transcriptomic studies, their limitations and especially their short read lengths make them poorly suited for some particular biological problems, including assembly, genome-wide determination of chromosome architecture, and genetic modification or engineering. Results We integrated Pacific Biosciences and Illumina sequencing platforms for the highest-quality genome assembly yet achieved, revealing seven telomere-to-telomere chromosomes (34,922,528 bp; 10877 genes) with 1630 newly predicted genes and >1.5 Mb of new sequences. Most new sequences are located on AT-rich blocks, including 7 centromeres, 14 subtelomeres, and 2329 interspersed AT-rich blocks. The seven QM6a centromeres separately consist of 24 conserved repeats and 37 putative centromere-encoded genes. These findings open up a new perspective for future centromere and chromosome architecture studies. Next, we demonstrate that sexual crossing readily induced cytosine-to-thymine point mutations on both tandem and unlinked duplicated sequences. We also show by bioinformatic analysis that T. reesei has evolved a robust repeat-induced point mutation (RIP) system to accumulate AT-rich sequences, with longer AT-rich blocks having more RIP mutations. The widespread distribution of AT-rich blocks correlates genome-wide partitions with gene clusters, explaining why clustering of genes has been reported to not influence gene expression in T. reesei. Conclusion Compartmentation of ancestral gene clusters by AT-rich blocks might promote flexibilities that are evolutionarily advantageous in this fungus’ soil habitats and other natural environments. Our analyses, together with the complete genome sequence, provide a better blueprint for biotechnological and industrial applications.

Lee, Shinyoung; Mo, Huaping; Kim, Jeong Im; Chapple, Clint

doi: 10.1186/s13068-017-0725-0pmid: 28239412

Abstract Background Monolignol-like molecules can be integrated into lignin along with conventional monolignol units, and it has been shown that the incorporation of non-canonical subunits can be used to generate hydrolysable lignin by introduction of ester linkages into the polymer and that this type of lignin is more easily removable. Disinapoyl esters (DSEs), which to some degree resemble the monolignol sinapyl alcohol, may be promising lignin modifying units for this purpose. As a first step toward determining whether this goal is achievable, we manipulated metabolic flux in Arabidopsis to increase the amounts of DSEs by overexpressing sinapoylglucose:sinapoylglucose sinapoyltransferase (SST) which produces two main DSEs, 1,2-disinapoylglucose, and another compound we identify in this report as 3,4-disinapoyl-fructopyranose. Results We succeeded in overproducing DSEs by introducing an SST-overexpression construct into the sinapoylglucose accumulator1 (sng1-6) mutant (SST-OE sng1-6) which lacks several of the enzymes that would otherwise compete for the SST substrate, sinapoyglucose. Introduction of cinnamyl alcohol dehydrogenase-c (cad-c) and cad-d mutations into the SST-OE sng1-6 line further increased DSEs. Surprisingly, a reduced epidermal fluorescence (ref) phenotype was observed when SST-OE sng1-6 plants were evaluated under UV light, which appears to have been induced by the sequestration of DSEs into subvacuolar compartments. Although we successfully upregulated the accumulation of the target DSEs, we did not find any evidence showing the integration of DSEs into the cell wall. Conclusions Our results suggest that although phenylpropanoid metabolic engineering is possible, a deeper understanding of sequestration and transport mechanisms will be necessary for successful lignin engineering through this route.

Shao, Yongni;Fang, Hui;Zhou, Hong;Wang, Qi;Zhu, Yiming;He, Yong

doi: 10.1186/s13068-017-0977-8pmid: 29255483

Abstract In this study, confocal Raman microspectroscopy was used to detect lipids in microalgae rapidly and non-destructively. Microalgae cells were cultured under nitrogen deficiency. The accumulation of lipids in Scenedesmus obliquus was observed by Nile red staining, and the total amount of lipids accumulated in the cells was measured by gravimetric method. The signals from different microalgae cells were collected by confocal Raman microspectroscopy to establish a prediction model of intracellular lipid content, and surface scanning signals for drawing pseudo color images of lipids distribution. The images can show the location of pyrenoid and lipid accumulation in cells. Analyze Raman spectrum data and build PCA-LDA model using four different bands (full bands, pigments, lipids, and mixed features). Models of full bands or pigment characteristic bands were capable of identifying S. obliquus cells under different nitrogen stress culture time. The prediction accuracy of model of lipid characteristic bands is relatively low. The correlation between the fatty acid content measured by the gravimetric method and the integral Raman intensity of the oil characteristic peak (1445 cm−1) measured by Raman spectroscopy was analyzed. There was significant correlation (R 2 = 0.83), which means that Raman spectroscopy is applicable to semi-quantitative detection of microalgal lipid content.

Ahmad, Qurat-ul-Ain; Yang, Shang-Tian; Manzoor, Maleeha; Qazi, Javed Iqbal

doi: 10.1186/s13068-017-0785-1pmid: 28450886

Abstract Background Biofuels obtained from first-generation (1G) sugars-starch streams have been proven unsustainable as their constant consumption is not only significantly costly for commercial scale production systems, but it could potentially lead to problems associated with extortionate food items for human usage. In this regard, biofuels’ production in alkali-thermophilic environs from second-generation (2G) bio-waste would not only be markedly feasible, but these extreme conditions might be able to sustain aseptic fermentations without spending much for sterilization. Results Present investigation deals with the valuation of ethanologenic potential of locally isolated moderate alkali-thermophilic fermentative bacterium, Bacillus licheniformis KU886221 employing sugarcane cane bagasse (SCB) as substrate. A standard 2-factor central composite response surface design was used to estimate the optimized cellulolytic and hemicellulolytic enzymatic hydrolysis of SCB into maximum fermentable sugars. After elucidation of optimized levels of fermentation factors affecting ethanol fermentation using Taguchi OA L27 (3^13) experimental design, free cell batch culture was carried out in bench-scale stirred-tank bioreactor for ethanol fermentation. Succeeding fermentation modifications included subsequent substrate addition, immobilized cells fibrous-bed bioreactor (FBB) incorporation to the basic setup, and performance of in situ gas stripping for attaining improved ethanol yield. Highest ethanol yield of 1.1406 mol ethanol/mol of equivalent sugars consumed was obtained when gas stripping was performed during fed-batch fermentation involving FBB under aseptic conditions. Despite the fact that under non-aseptic conditions, 30.5% lesser ethanol was formed, still, reduced yield might be considered influential as it saved the cost of sterilization for ethanol production. Conclusion Effectual utilization of low-priced abundantly available lignocellulosic waste sugarcane bagasse under non-aseptic moderate alkali-thermophilic fermentation conditions as directed in this study has appeared very promising for large-scale cost-effective bioethanol generation processes.