Microaerobic fermentation of algae biomass for increased liquid fuels yield and nutrient recycling.
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A method for producing one or more hydrocarbon compounds from at least one of 2,3-butanediol, acetoin, and ethanol, the method comprising contacting said at least one of 2,3-butanediol, acetoin, and ethanol with a catalyst at a temperature of at least 100° C. and up to 500° C. to result in said 2,3-butanediol, acetoin, and/or ethanol being converted to said one or more hydrocarbon compounds, wherein said catalyst is either: (i) a catalyst comprising nanoparticles composed of (a) a first metal oxide selected from the group consisting of zirconium oxide, cerium oxide, titanium oxide, and lanthanum oxide, and (b) a main group metal oxide; or (ii) a catalyst comprising a zeolite loaded with at least one metal selected from the group consisting of copper, silver, nickel, palladium, platinum, rhodium, and ruthenium in an amount of 1-30 wt % by weight of the zeolite.
A method of preparing a product derived from a rhamnolipid includes the steps of: providing a rhamnolipid, combining the rhamnolipid with a reagent, allowing the rhamnolipid and reagent to react to form a product derived from the rhamnolipid, and collecting the product derived from the rhamnolipid. An exemplary product is dimeric β-hydroxy fatty acid.
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A process for the preparation of bio-based malonic acid and crystalline calcium malonate is provided. The calcium malonate is highly pure and provides a source of malonic acid made from a renewable carbon source rather than existing processes which rely on the use of petroleum-based products. The calcium malonate provides an improved source of malonic acid, which is important to many industrial processes.
The present invention provides an isolated AZF1 gene sequence, recombinant vectors, and recombinant yeast which are useful in methods of enhanced biofuel production, particularly ethanol production. Methods of bioengineering recombinant yeast with isolated AZF1 gene sequence useful for biofuel production are also provided.
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Background: The fermentation inhibition of yeast or bacteria by lignocellulose-derived degradation products, during hexose/pentose co-fermentation, is a major bottleneck for cost-effective lignocellulosic biorefineries. To engineer microbial strains for improved performance, it is critical to understand the mechanisms of inhibition that affect fermentative organisms in the presence of major components of a lignocellulosic hydrolysate. The development of a synthetic lignocellulosic hydrolysate (SH) media with a composition similar to the actual biomass hydrolysate will be an important advancement to facilitate these studies. In this work, we characterized the nutrients and plant-derived decomposition products present in AFEX™ pretreated corn stover hydrolysate (ACH). The SH was formulated based on the ACH composition and was further used to evaluate the inhibitory effects of various families of decomposition products during Saccharomyces cerevisiae 424A (LNH-ST) fermentation. Results: The ACH contained high levels of nitrogenous compounds, notably amides, pyrazines, and imidazoles. In contrast, a relatively low content of furans and aromatic and aliphatic acids were found in the ACH. Though most of the families of decomposition products were inhibitory to xylose fermentation, due to their abundance, the nitrogenous compounds showed the most inhibition. From these compounds, amides (products of the ammonolysis reaction) contributed the most to the reduction of the fermentation performance. However, this result is associated to a concentration effect, as the corresponding carboxylic acids (products of hydrolysis) promoted greater inhibition when present at the same molar concentration as the amides. Due to its complexity, the formulated SH did not perfectly match the fermentation profile of the actual hydrolysate, especially the growth curve. However, the SH formulation was effective for studying the inhibitory effect of various compounds on yeast fermentation. Conclusions: The formulation of SHs is an important advancement for future multi-omics studies and for better understanding the mechanisms of fermentation inhibition in lignocellulosic hydrolysates. The SH formulated in this work was instrumental for defining the most important inhibitors in the ACH. Major AFEX decomposition products are less inhibitory to yeast fermentation than the products of dilute acid or steam explosion pretreatments; thus, ACH is readily fermentable by yeast without any detoxification.
Bench Scale Integration develops and optimizes fermentation processes to produce bio-based fuels and chemicals for commercial scale-up. The project uses fermentation science to achieve high titers and production rates by, for example, manipulating how the microorganisms are fed biomass sugars and nutrients, modifying fermentation conditions (pH, temperature, aeration) or developing online control strategies for better fermentation operations and high titer, rates, and yield (TRY). For this period of performance, we continued our development of a commercial-ready 2,3-butanediol (BDO) fermentation from biomass sugars utilizing NREL's proprietary Zymomonas mobilis microorganism. The engineered Z. mobilis can use all the main sugars in corn stover biomass, which are glucose, xylose, and arabinose. BDO is a versatile, low-carbon chemical which can be catalytically upgraded to a variety of hydrocarbon fuels and chemicals. The project had three goals during this review period; evaluate the technical feasibility of using whole slurry pretreated corn stover to achieve the techno-economic analysis (TEA) performance goals, continue optimizing a liquor-based fed-batch fermentation process for high titer, and develop strategies to enable scale-up. After evaluating different iterations of a whole slurry fermentation that did not meet the TEA goals, a Go/No-Go decision was made to pivot to liquor-only with new TEA performance targets, the main one being 140 g/L titer. We successfully met this goal, producing 141 g/L BDO at 1 g/L-hr productivity and 84% process yield. This titer and productivity attracted industrial interest to scale the fermentation resulting in a Technology Commercialization Fund project award in 2022. We used an NREL developed near-infra red (NIR) spectroscopy method for rapid analysis which allowed for changes to aeration levels and sugar feeding during the fermentation to maximize BDO production. The NIR analysis can be done using a hand-held spectrometer, essentially taking the analysis on to the plant floor during scale-up and preliminary work shows the feasibility of using an online probe for continuous monitoring and control. The other scale-up tool is mapping oxygen transfer coefficient (kLa) and oxygen transfer rate (OTR) in various vessels to find conditions that match the optimized 500 mL vessels. Showing a correlation to the mapping work, which is done with water and a dissolved oxygen probe, can reduce the risk of failed fermentations during scale-up. Our end-of-project goal is to meet the design target BDO titer (140-150 g/L) from DMR corn stover liquor at 1000L or larger scale to demonstrate BDO process design case and transfer the technology to industrial fermentation stakeholders for commercialization.
Butyric acid is a valuable chemical that can be fermentatively produced in Clostridium tyrobutyricum and catalytically upgraded to drop in biofuels. Despite considerable study of glucose fermentation in C. tyrobutyricum, information about glucose and xylose co-fermentation to produce butyric acid is limited. In this work, fed-batch fermentations of corn stover hydrolysate at pH 5.0 demonstrated the co-fermentation of glucose and xylose. However, the fermentation stalled at approximately 48 hours after reaching inhibitory levels of butyric acid (18 g/L). When the fermentation pH was increased to 6.0, higher butyric acid titers (50 g/L) were achieved but the fermentation again stalled after 98 hours due to product inhibition. To demonstrate the potential of a continuous co-fermentation process, in situ product recovery via membrane-based liquid-liquid extraction (pertraction) was applied to remove butyric acid. The extracted acids were then recovered from the organic solvent via back extraction into an alkaline solution, producing a high purity product. Results from pertractive fermentations of real corn stover hydrolysate are shown below.
Sewage sludge was taken from a dairy WWTP belonging to Mlekovita Cooperative in Wysokie Mazowieckie. There were excess sludge, flotation sludge and a mixture of excess and flotation sludge from pre-treatment of dairy sewage. The initial content of 16 PAHs in excess sludge before fermentation was approximately 689 µg·kg{sup −1} in dry mass, whereas in post-flotation sludge (which constituted around 30% of raw sludge) it was approximately 95 µg·kg{sup −1} in dry mass. A mixture of excess and flotation sludge had the content of 497,7 µg·kg{sup −1} in dry mass. Through comparison of particular hydrocarbons content in raw sewage sludge to the total PAHs content, it was shown that tricyclic compounds, which constituted 46,3% of the PAHs sum (excess sludge), and tetracyclic compounds, which constituted 60,0% of the PAHs sum (flotation sludge), were the dominating fractions. In the sludge subjected to fermentation in reactors with mixed sludge and surplus activated sludge, the general trend of the course of changes in concentrations of PAHs was similar. Both in the sludge inoculated with EM and in that not inoculated with EM, a significant increase in the total PAHs contents was observed in the first fermentation phase (acidic fermentation) after 7 days of the process. Addition of EM into the sludge did not prevent the PAHs release, and therefore higher concentrations of PAHs sum were recorded during the hydrolysis stage than in sludge before fermentation. A decrease in the sum of PAHs was observed after 2 weeks of fermentation in relation to the quantity observed after 1 week of fermentation (except from post-flotation sludge). In the following weeks, there was further decrease in the concentration of the 16 PAHs sum in all sludge types. However, in sludge without EM inoculation, it was lower than in sludge with EM inoculation. The loss of the majority of tested hydrocarbons was reported in the final phase of fermentation. - Highlights: • The influence of applying Effective Microorganisms on PAHs degradation in dairy sewage sludge during fermentation has been examined. • To study the relationship between quantitative parameters and to describe the correlation strength, Spearman's rank correlation coefficient was used. • In order to verify whether there was a statistically significant change, the Wilcoxon test was applied for individual observation pairs.
Abstract Background Environmental factors, such as weather extremes, have the potential to cause adverse effects on plant biomass quality and quantity. Beyond adversely affecting feedstock yield and composition, which have been extensively studied, environmental factors can have detrimental effects on saccharification and fermentation processes in biofuel production. Only a few studies have evaluated the effect of these factors on biomass deconstruction into biofuel and resulting fuel yields. This field-to-fuel evaluation of various feedstocks requires rigorous coordination of pretreatment, enzymatic hydrolysis, and fermentation experiments. A large number of biomass samples, often in limited quantity, are needed to thoroughly understand the effect of environmental conditions on biofuel production. This requires greater processing and analytical throughput of industrially relevant, high solids loading hydrolysates for fermentation, and led to the need for a laboratory-scale high solids experimentation platform. Results A field-to-fuel platform was developed to provide sufficient volumes of high solids loading enzymatic hydrolysate for fermentation. AFEX pretreatment was conducted in custom pretreatment reactors, followed by high solids enzymatic hydrolysis. To accommodate enzymatic hydrolysis of multiple samples, roller bottles were used to overcome the bottlenecks of mixing and reduced sugar yields at high solids loading, while allowing greater sample throughput than possible in bioreactors. The roller bottle method provided 42–47% greater liquefaction compared to the batch shake flask method for the same solids loading. In fermentation experiments, hydrolysates from roller bottles were fermented more rapidly, with greater xylose consumption, but lower final ethanol yields and CO 2 production than hydrolysates generated with shake flasks. The entire platform was tested and was able to replicate patterns of fermentation inhibition previously observed for experiments conducted in larger-scale reactors and bioreactors, showing divergent fermentation patterns for drought and normal year switchgrass hydrolysates. Conclusion A pipeline of small-scale AFEX pretreatment and roller bottle enzymatic hydrolysis was able to provide adequate quantities of hydrolysate for respirometer fermentation experiments and was able to overcome hydrolysis bottlenecks at high solids loading by obtaining greater liquefaction compared to batch shake flask hydrolysis. Thus, the roller bottle method can be effectively utilized to compare divergent feedstocks and diverse process conditions.
The Value Prior to Pulping (VPP) project goal was to demonstrate the technical and commercial feasibility of introducing a new value stream into existing pulp and paper mills. Essentially the intent was to transfer the energy content of extracted hemicellulose from electricity and steam generated in the recovery boiler to a liquid transportation fuel. The hemicellulose fraction was extractedprior to pulping, fractionated, or conditioned if necessary, and fermented to ethanol. Commercial adaptation of the process to wood hemicelluloses was a prerequisite for using this less currently valued component available from biomass and wood. These hemicelluloses are predominately glucurono-xylan in hardwoods and galactoglucomannan in softwoods (with a significant softwood component of anarabino-xylan) and will yield fermentation substrates different from cellulose. NREL provided its expertise in the area of fermentation host evaluation using its Zymomonas strains on the CleanTech Partner's (CTP) VPP project. The project was focused on the production of fuel ethanol and acetic acid from hemicellulose streams generated from wood chips of industrially important hardwood andsoftwood species. NREL was one of four partners whose ethanologen was tested on the hydrolyzed extracts. The use of commercially available enzymes to treat oligomeric sugar extracts was also investigated and coupled with fermentation. Fermentations by NREL were conducted with the Zymomonas mobilis organism with most of the work being performed with the 8b strain. The wood extracts hydrolyzedand/or fermented by NREL were those derived from maple, mixed southern hardwoods, and loblolly pine. An unhydrolyzed variant of the mixed southern hardwood extract possessed a large concentration of oligomeric sugars and enzymatic hydrolysis was performed with a number of enzymes, followed by fermentation. The fermentation of the wood extracts was carried out at bench scale in flasks or smallbioreactors, with a maximum volume of 500 mL.
Microbial production of succinic acid (SA) at an industrially relevant scale has been hindered by high downstream processing costs arising from neutral pH fermentation for over three decades. Here, we metabolically engineer the acid-tolerant yeast Issatchenkia orientalis for SA production, attaining the highest titers in sugar-based media at low pH (pH 3) in fed-batch fermentations, i.e. 109.5 g/L in minimal medium and 104.6 g/L in sugarcane juice medium. We further perform batch fermentation using sugarcane juice medium in a pilot-scale fermenter (300×) and achieve 63.1 g/L of SA, which can be directly crystallized with a yield of 64.0%. Finally, we simulate an end-to-end low-pH SA production pipeline, and techno-economic analysis and life cycle assessment indicate our process is financially viable and can reduce greenhouse gas emissions by 34–90% relative to fossil-based production processes. We expect I. orientalis can serve as a general industrial platform for production of organic acids.
Rising concerns for sustainability and global climate change have driven the development of sustainable production pathways for biofuels and chemicals from lignocellulosic biomass via integrated biological and chemical processes. We constructed an engineered Saccharomyces cerevisiae capable of producing 2,3-butanediol (2,3-BDO) from glucose without accumulating ethanol and glycerol, which hinder downstream processing of 2,3-BDO, through extensive metabolic reprogramming. Specifically, we introduced heterologous 2,3-BDO biosynthetic enzymes and deleted the major isozymes of ethanol and glycerol biosynthetic enzymes. In addition, we introduced an NAD+ regenerating Pyruvate-Malate (PM) cycle and enhanced the NAD+ regenerating capability of the PM cycle to resolve the redox imbalance from the deletion of ethanol and glycerol production pathways. The resulting engineered yeast produced 109.9 g/L of 2,3-BDO with a productivity of 1.0 g/L/h and a yield of 0.36 g/g glucose in a fed-batch fermentation. We also conducted techno-economic analysis (TEA) and life cycle assessment (LCA) of the production of methyl ethyl ketone (MEK) through catalytic dehydration of 2,3-BDO. A TEA based on the experimental results indicated that the minimum product selling price (MPSP) was estimated to be $1.90/kg. Regarding cradle-to-grave LCA, 100-year global warming potential (GWP100) and fossil energy consumption (FEC) were found to be 0.37 kg CO2 eq/kg and 3.1 MJ/kg, respectively. These results demonstrated the feasibility of cost-competitive and sustainable bio-based MEK production via yeast fermentation. In addition, we explored the possibility of using the fermentation broth containing 2,3-BDO as a biostimulant inducing drought tolerance in plants. As a result, the yeast 2,3-BDO fermentation broth can induce drought tolerance in Arabidopsis thaliana without a complicated purification process.
Efforts to engineer high-productivity crops to accumulate oils in their vegetative tissue present the possibility of expanding biodiesel production. However, processing the new crops for lipid recovery and ethanol production from cell wall saccharides is challenging and expensive. In a previous study using corn germ meal as a model substrate, we reported that liquid hot water (LHW) pretreatment enriched the lipid concentration by 2.2 to 4.2 fold. This study investigated combining oil recovery with ethanol production by extracting oil following LHW and simultaneous saccharification and co-fermentation (SSCF) of the biomass. Corn germ meal was again used to model the oil-bearing energy crops. Pretreated germ meal hydrolysate or solids (160 °C and 180 °C for 10 minutes) were fermented, and lipids were extracted from both the spent fermentation whole broth and fermentation solids, which were recovered by centrifugation and convective drying. Lipid contents in spent fermentation solids increased 3.7 to 5.7 fold compared to the beginning germ meal. The highest lipid yield achieved after fermentation was 36.0 mg lipid g−1 raw biomass; the maximum relative amount of triacylglycerol (TAG) was 50.9% of extracted oil. Although the fermentation step increased the lipid concentration of the recovered solids, it did not improve the lipid yields of pretreated biomass and detrimentally affected oil compositions by increasing the relative concentrations of free fatty acids.