Fermentation of glycerol and production of valuable chemical and biofuel molecules

Glycerol has attracted the attention of scientific and industrial communities due to its generation in bulk quantities as a byproduct of biofuel industries. With the rapid growth of these industries in recent years, glycerol is frequently treated as a very low-value byproduct or even a waste product with a disposal cost associated to it. Glycerol is not only abundant and inexpensive but also can generate more reducing equivalents than glucose or xylose. This unique characteristic of glycerol offers a tremendous opportunity for its biological conversion to valuable products at higher yield. This review focuses on research efforts to utilize glycerol as a carbon source for the production of a variety of fuels and chemicals by both native and metabolically engineered microorganisms.     

Optimization of Chlorella biomass harvesting by flocculation and its potential for biofuel production

Optimization of microalgal biomass harvesting is essential to produce effective and optimum outcomes that can contribute towards a feasible and economical harvesting technique. Two Chlorella species were used, namely, C. vulgaris and C. sorokiniana UKM3. Two essential factors affecting microalgal biomass harvesting via flocculation, namely, the initial pH of the microalgal broth and flocculant (chitosan) concentration were studied. The optimization process was conducted by using a response surface methodology (RSM) based on the model of face-centered-central composite design (FC-CCD). The potential for biofuel application of the harvested biomass was evaluated based on the production of fatty acid methyl esters (FAMEs) by transesterification. The quadratic models obtained from the RSM significantly fitted the experiment data as the p-values were less than 0.05. The initial pH of the microalgal suspension was found to have a more significant effect on the flocculation process than flocculant concentration. For C. vulgaris, the highest flocculant efficiency of 98.7% was obtained at a chitosan concentration of 0.2 g L^?1 and an initial pH of 12.0, whereas for C. sorokiniana UKM3, at 0.15 g L^?1 of chitosan and initial pH of 12.0 produced the highest efficiency of 97.1%. The harvested biomass of both species exhibited a high content of palmitic acid (C16:0) with 29.74 wt% and 11.81 wt% of dry biomass for C. vulgaris and C. sorokiniana UKM3, respectively. This study showed that Chlorella species can be harvested efficiently using the flocculation process and manifested an excellent potential for biodiesel production where palmitic acid (C16:0) is one of the main compounds for high-acid oil-biodiesel.

     

Lipid biofuel production with microalgae: potential and challenges

Microalgae can accumulate fatty acids up to 80% of their dry weight (Chisti 2007). As a consequence the yield per hectare could be higher by a factor of 30 compared to terrestrial oleaginous species. Biodiversity of microalgae is enormous. It is estimated that there are between 200 000 and several million species. Such diversity is an unexplored potential for research and industry. In comparison to terrestrial oleaginous species, microalgae have many characteristics addressing environmental problems with a drastically enhanced oil production. Microalgae are currently generating mediatic enthusiasm, and many start-ups are investing this niche. Nevertheless there are still locks to undo via upstream search, before the expected returns and costs are met and before these technologies can be developed at a large scale.     

Fermentation of glycerol to 1,3-propanediol: use of cosubstrates

Three fermentable substances, glucose, 1,2-ethanediol and 1,2-propanediol were checked as cosubstrates for the fermentation of glycerol by Clostridium butyricum and Citrobacter freundii with the aim of achieving a complete conversion of glycerol to 1,3-propanediol. Glucose was fermented by C. butyricum mainly to acetate, CO₂ and reducing equivalents in the presence of glycerol and contributed markedly to the 1,3-propanediol yield. However, because of relatively slow growth on glucose, complete conversion was not achieved. If the two glycols were used as cosubstrates for glycerol fermentation, the 1,3-propanediol yield did not increase but dimished considerably, as they were converted to more reduced products, i.e. alcohols instead of acids. From 1,2-propanediol 2-propanol was formed in addition to 1-propanol. The ratio of the propanols was dependent on the culture conditions.     

The potential of sustainable algal biofuel production using wastewater resources

The potential of microalgae as a source of renewable energy has received considerable interest, but if microalgal biofuel production is to be economically viable and sustainable, further optimization of mass culture conditions are needed. Wastewaters derived from municipal, agricultural and industrial activities potentially provide cost-effective and sustainable means of algal growth for biofuels. In addition, there is also potential for combining wastewater treatment by algae, such as nutrient removal, with biofuel production. Here we will review the current research on this topic and discuss the potential benefits and limitations of using wastewaters as resources for cost-effective microalgal biofuel production.     

Exploring potential use of Australian thraustochytrids for the bioconversion of glycerol to omega-3 and carotenoids production

Marine microbes have the potential for accumulating large quantities of lipids and are therefore suitable candidate as feedstock in unsaturated fatty acid production. The efficient utilisation of glycerol as an alternative carbon source to glucose was demonstrated in the fermentation of newly isolated thraustochytrid strains from the Queenscliff, Victoria, Australia. The isolates exhibited the presence of omega-3 and omega-6 polyunsaturated fatty acids, with the major fatty acids for all isolates being (as percent total fatty acid), palmitic acid (25.1–40.78%), stearic acid (4.24–13.2%), eicosapentaenoic acid EPA (2.31–8.5%) and docosapentaenoic acid (7.24–10.9%). Glycerol as a carbon source gave promising biomass growth with significant lipid and DHA productivity. An approximate three-fold increase in carotenoid content in all isolates was achieved when glycerol was used as a carbon source in the production medium.     

Fermentation of biodiesel-derived glycerol by Bacillus amyloliquefaciens: effects of co-substrates on 2,3-butanediol production

Cultivation in glycerol instead of sugars inhibits 2,3-butanediol (2,3-BD) production by Bacillus amyloliquefaciens. In this study, we report that B. amyloliquefaciens readily produces 2,3-BD from biodiesel-derived glycerol in the presence of beet molasses as a co-substrate. Unexpectedly, the molasses stimulated 2,3-BD production and simultaneously reduced the duration of fermentation. Productivity of 2,3-BD was enhanced at the start of fermentation, and yields increased under continuous molasses supply. Subsequently, 2,3-BD production in molasses-supplemented fed-batch culture was observed. Prior to inoculation of fed-batch fermentation culture, 15 g/l of molasses was added to the bioreactor. After 6 h of incubation, the bioreactor was fed with a solution containing 80 % glycerol and 15 % molasses. The 2,3-BD concentration, yield, and productivity significantly improved, reaching 83.3 g/l, 0.42 g/g, and 0.87 g/l·h, respectively. To our knowledge, these results are the highest report for 2,3-BD fermentation from biodiesel-derived glycerol.     

Macroalgae in biofuel production

The conversion processes of macroalgae for biofuels can be divided into thermochemical (dry) and microbiological (wet) processes. The chemical composition of macroalgae together with the pre‐treatment method, conversion conditions, and the characteristics of the microbes involved (wet processes) determine the yield and the properties of the biofuel produced. Macroalgae are often rich in carbohydrates, and therefore well suited for biogas, biobutanol and bioethanol productions. The content of triacylglycerols (TAGs) is the best indicator for the suitability of the alga for biodiesel production. TAGs have a high conversion rate to biodiesel, high percentage of fatty acids, and they lack phosphorus, sulfur and nitrogen. Macroalgae can have high metal concentrations, which can have an impact on conversion processes: metals may inhibit or catalyse the processes. High sulfur (especially in green algae) and nitrogen contents are also characteristic to macroalgae, and may be problematic in the production of biogas (NH₃‐toxicity) and the use of the oil and biodiesel (high concentrations of H₂S and NO_x‐compounds). Macroalgae have proven to be suitable material for conversion processes, but further optimization of the processes is needed. At present, macroalgae are not economically, or in many cases not even environmentally, sustainable material when the whole production chain is considered. In this review we summarize information on the chemical composition of macroalgae in a prospect of biofuel production, and the current situation in the field of macroalgal‐based biofuel production.     

Fermentation of glycerol by Clostridium pasteurianum--batch and continuous culture studies

The fermentation of glycerol by Clostridium pasteurianum was studied with respect to product formation as influenced by the culture conditions. In the majority of batch cultures, butanol was the main fermentation product, but a varying fraction of glycerol was also converted to 1,3-propanediol, butyric and acetic acids and ethanol. More than 60 g/l glycerol was utilized, and up to 17 g/l butanol was produced. Fed-batch cultures did not offer an advantage. When molecular nitrogen was used as a nitrogen source, the fermentation time was prolonged by a factor of 1.5. Fermentations at constant pH values between 4.5 and 7.5 did not reveal significant differences in product formation except for an increase in the ethanol content starting at pH 6.5. Chemostat cultures also yielded predominantly n-butanol, but in some fermentations, the 1,3-propanediol fraction was relatively high. The pH auxostat cultures, which were operated at a glycerol excess, contained 1,3-propanediol as the main product. As a whole, the fermentations were characterized by a certain variability in product formation under seemingly equal or slightly varied conditions. It appears that the regulation of the numerous fermentation pathways occurring in this organism is not very strict. Journal of Industrial Microbiology & Biotechnology (2001) 27, 18–26. Received 25 September 2000/ Accepted in revised form 07 April 2001     

Advanced biofuel production in microbes

The cost-effective production of biofuels from renewable materials will begin to address energy security and climate change concerns. Ethanol, naturally produced by microorganisms, is currently the major biofuel in the transportation sector. However, its low energy content and incompatibility with existing fuel distribution and storage infrastructure limits its economic use in the future. Advanced biofuels, such as long chain alcohols and isoprenoid- and fatty acid-based biofuels, have physical properties that more closely resemble petroleum-derived fuels, and as such are an attractive alternative for the future supplementation or replacement of petroleum-derived fuels. Here, we review recent developments in the engineering of metabolic pathways for the production of known and potential advanced biofuels by microorganisms. We concentrate on the metabolic engineering of genetically tractable organisms such as Escherichia coli and Saccharomyces cerevisiae for the production of these advanced biofuels.     

Fermentation production ofL-Homoserine byCorynehacterium sp. and its possible use in the preparation of threonine and lysine

A mutant Corynebacterium sp. requiring threonine and cultivated for 3 d in a medium containing 15% sucrose, 8% corn-steep and 50 micrograms biotin per litre accumulated 14.5 g L-homoserine per litre. The possibility of fermenting the homoserine obtained for threonine and lysine production was investigated.     

Mineral concentration in selected native temperate grasses with potential use as biofuel feedstock

Stands of native grasses along roadways, in buffer strips, riparian zones and grass prairies have potential utility as feedstock for bioenergy production. The sustainability of harvesting these stands is reliant, in part, on knowledge of the mineral concentration of the harvested grasses because removal of mineral nutrients such as phosphorus (P) and potassium (K) can impact subsequent biomass production and ecosystem services associated with these stands. Mineral content of biomass, particularly that of silicon (Si), chlorine (Cl), and sulfur (S) also impacts thermochemical conversion approaches that convert grasses into bioproducts. This study quantified Cl, S, Si, P and K in Bromus marginatus, Elymus glaucus, Poa secunda, Pseudoroegneria, Elymus lanceolatus, Elymus trachycaulus, Leymus cinereus, Leymus triticoides, and Pseudoroegneria spicata collected at three growth developmental stages from four plant introduction stations located in the western US. Differences (P ? 0.05) in mineral concentrations were associated with developmental stage, species, and location. Variability was greatest in Si concentrations which ranged from 1847 to 28620 mg kg⁻¹, similar to those recorded in other grasses. Variability in Cl and S concentrations also occurred, but at less magnitude than that of Si. Concentrations of P and K, two mineral fertilizer components, varied approximately threefold among these grasses. Differences in mineral concentrations among these grasses were not completely dependent upon soil mineral content. Long-term evaluations of available soil mineral concentrations under contrasting management practices are needed to quantify how local conditions impact mineral cycling, and in turn, the sustainability of harvesting these stands. The data presented here establish baselines for these species in locations subject to contrasting environmental and microbiological conditions that affect mineral cycling and availability.     

Regional examination shows potential for native feedstock options for cellulosic biofuel production

Kentucky, as with many regions around the globe, has a relatively long growing season with significant rainfall that could produce sizeable quantities of perennial herbaceous and woody biomass on land that does not compete with food crops. Additionally, there are limited options for renewable power production from low carbon sources such as solar-photovoltaic, wind and hydroelectric. Recent studies have shown that producing renewable energy from perennial cellulosic crops, as opposed to starch-based biofuel crops, will have a carbon-mitigating outcome. Currently, there is a lack of data regarding regionally suitable genotypes. Herein, we establish baseline values for multiple entry selections of three native C4 grass species, switchgrass (SW) ( Panicum virgatum L.), eastern gamagrass (EG) (Trispicum dactyloides L. ) and big bluestem (BB) (Andropogon gerardii Vitman ) . Yield potential examined over 7 years showed that environment, species and entries had a significant impact on yield, but EG had higher total yield over the duration of the study. Cellulosic biofuel potential was examined by measurement of saccharification efficiency, relative lignocellulosic energy density, cellulose content and lignin content during three growing seasons. EG had significantly higher digestibility rate than SW and BB. Underlying this was a negative correlation between lignification and saccharification efficiency. However, higher lignin content and higher cellulose content among SW entries resulted in higher energy density relative to EG and BB. These data reveal that locally bred EG varieties were most suited to cellulosic ethanol production under the growing conditions of central Kentucky, USA, compared with SW and BB and suggest the importance of regional examination.     

Advanced biofuel production by the yeast Saccharomyces cerevisiae

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Fermentation of glycerol to 1,3-propanediol by Klebsiella and Citrobacter strains

Summary Glycerol-fermenting anaerobes were enriched with glycerol at low and high concentrations in order to obtain strains that produce 1,3-propanediol. Six isolates were selected for more detailed characterization; four of them were identified as Citrobacter freundii, one as Klebsiella oxytoca and one as K. pneumoniae. The Citrobacter strains formed 1.3-propanediol and acetate and almost no by-products, while the Klebsiella strains produced varying amounts of ethanol in addition and accordingly less 1,3-propanediol. Enterobacterial strains of the genera Enterobacter, Klebsiella, and Citrobacter from culture collections showed similar product patterns except for one group which formed limited amounts of ethanol, but no propanediol. Seven strains were grown in pH-controlled batch cultures to determine the parameters necessary to evaluate their capacity for 1,3-propanediol production. K. pneumoniae DSM 2026 exhibited the highest final concentration (61 g/l) and the best productivity (1.7 g/l h) whereas C. freundii Zu and K2 achieved only 35 g/l and 1.4 g/l h, respectively. The Citrobacter strains on the other hand gave somewhat better yields which were very close to the theoretical optimum of 65 mol %. Offprint requests to: H. Biebl     

Potential of biofilm-based biofuel production

Biofilm technology has been extensively applied to wastewater treatment, but its potential application in biofuel production has not been explored. Current technologies of converting lignocellulose materials to biofuel are hampered by costly processing steps in pretreatment, saccharification, and product recovery. Biofilms may have a potential to improve efficiency of these processes. Advantages of biofilms include concentration of cell-associated hydrolytic enzymes at the biofilm–substrate interface to increase reaction rates, a layered microbial structure in which multiple species may sequentially convert complex substrates and coferment hexose and pentose as hydrolysates diffuse outward, and the possibility of fungal–bacterial symbioses that allow simultaneous delignification and saccharification. More importantly, the confined microenvironment within a biofilm selectively rewards cells with better phenotypes conferred from intercellular gene or signal exchange, a process which is absent in suspended cultures. The immobilized property of biofilm, especially when affixed to a membrane, simplifies the separation of biofuel from its producer and promotes retention of biomass for continued reaction in the fermenter. Highly consolidated bioprocessing, including delignification, saccharification, fermentation, and separation in a single reactor, may be possible through the application of biofilm technology. To date, solid-state fermentation is the only biofuel process to which the advantages of biofilms have been applied, even though it has received limited attention and improvements. The transfer of biofilm technology from environmental engineering has the potential to spur great innovations in the optimization of biofuel production.