An alternative biorefinery approach to address microalgal seasonality:Blending with spent coffee grounds

An effective method for the production of fuels and chemicals from microalgae is to ferment the carbohydrate fraction, extract the lipids and convert the resulting solids through hydrothermal liquefaction (HTL). In this process, known as Combined Algal Processing (CAP), multiple fuel precursors are produced effectively. However, one of the key challenges associated with a microalgae-based biorefinery is the reduced productivity of algae in the colder seasons. In this investigation, the potential for spent coffee grounds (SCG), a potentially valuable waste stream, to be blended with biomass from the microalgae Scenedesmus acutus (HCSD) to make up for the productivity shortfalls in periods of lower microalgae productivity to maximize the capacity for downstream equipment throughout the year was evaluated. Two different blend ratios were compared to only microalgae biomass or SCG, one representing winter season (40% microalgae and 60% SCG-blend 1) and another representing autumn and early spring (60% microalgae and 40% SCG-blend 2). Pretreatment of the blends showed higher monosaccharide release yields compared to microalgae alone, with an increase in mannose and galactose specifically. In the fermentation of the pretreated slurries, all the monosaccharides were consumed, resulting in ethanol titers of up to 23 g L-1 for the SCG blend, compared to 14 g L-1 ethanol for the algae alone. The lipid extraction from the blends resulted in yields of 95.5-99.7% (which translates to 173.8-193.5 kg per tonne of dry biomass processed in this biorefinery scenario) compared to 92.2% in HCSD (216.2 kg per tonne of dry biomass) and 68.1% in SCG (90.8 kg per tonne of dry biomass) alone. The residual solids left after fermentation and lipid extraction were converted via hydrothermal liquefaction (HTL) to produce bio-crude. The bio-crude yield was higher for microalgae (24.6%) than for the two blend cases (blend 1-17.5% and blend 2-19.7%). Theoretical energy calculations showed that the addition of SCG gave similar yields of fuel (gallon of gasoline equivalents) from the blends when compared to microalgae alone (94.7-96.5% depending on the blend of SCG). This work demonstrates that SCG can be easily incorporated with microalgae into a combined processing methodology and can therefore be used effectively during periods of lower availability of microalgae maintaining maximum operating levels of the conversion process equipment year-round. Moreover, co-processing algae with SCG not only leads to increased ethanol titers in the fermentation but also improves the lipid extraction yields. This journal is </p

Ameliorating the Metabolic Burden of the Co-expression of Secreted Fungal Cellulases in a High Lipid-Accumulating Yarrowia lipolytica Strain by Medium C/N Ratio and a Chemical Chaperone

Yarrowia lipolytica, known to accumulate lipids intracellularly, lacks the cellulolytic enzymes needed to break down solid biomass directly. This study aimed to evaluate the potential metabolic burden of expressing core cellulolytic enzymes in an engineered high lipid-accumulating strain of Y. lipolytica. Three fungal cellulases, Talaromyces emersonii-Trichoderma reesei chimeric cellobiohydrolase I (chimeric-CBH I), T. reesei cellobiohydrolase II (CBH II), and T. reesei endoglucanase II (EG II) were expressed using three constitutive strong promoters as a single integrative expression block in a recently engineered lipid hyper-accumulating strain of Y. lipolytica (HA1). In yeast extract-peptone-dextrose (YPD) medium, the resulting cellulase co-expressing transformant YL165-1 had the chimeric-CBH I, CBH II, and EG II secretion titers being 26, 17, and 132 mg L-1, respectively. Cellulase co-expression in YL165-1 in culture media with a moderate C/N ratio of ∼4.5 unexpectedly resulted in a nearly two-fold reduction in cellular lipid accumulation compared to the parental control strain, a sign of cellular metabolic drain. Such metabolic drain was ameliorated when grown in media with a high C/N ratio of 59 having a higher glucose utilization rate that led to approximately twofold more cell mass and threefold more lipid production per liter culture compared to parental control strain, suggesting cross-talk between cellulase and lipid production, both of which involve the endoplasmic reticulum (ER). Most importantly, we found that the chemical chaperone, trimethylamine N-oxide dihydride increased glucose utilization, cell mass and total lipid titer in the transformants, suggesting further amelioration of the metabolic drain. This is the first study examining lipid production in cellulase-expressing Y. lipolytica strains under various C/N ratio media and with a chemical chaperone highlighting the metabolic complexity for developing robust, cellulolytic and lipogenic yeast strains

Transcriptional Analysis of Lactobacillus brevis to N-Butanol and Ferulic Acid Stress Responses

The presence of anti-microbial phenolic compounds, such as the model compound ferulic acid, in biomass hydrolysates pose significant challenges to the widespread use of biomass in conjunction with whole cell biocatalysis or fermentation. Currently, these inhibitory compounds must be removed through additional downstream processing or sufficiently diluted to create environments suitable for most industrially important microbial strains. Simultaneously, product toxicity must also be overcome to allow for efficient production of next generation biofuels such as n-butanol, isopropanol, and others from these low cost feedstocks.This study explores the high ferulic acid and n-butanol tolerance in Lactobacillus brevis, a lactic acid bacterium often found in fermentation processes, by global transcriptional response analysis. The transcriptional profile of L. brevis reveals that the presence of ferulic acid triggers the expression of currently uncharacterized membrane proteins, possibly in an effort to counteract ferulic acid induced changes in membrane fluidity and ion leakage. In contrast to the ferulic acid stress response, n-butanol challenges to growing cultures primarily induce genes within the fatty acid synthesis pathway and reduced the proportion of 19:1 cyclopropane fatty acid within the L. brevis membrane. Both inhibitors also triggered generalized stress responses. Separate attempts to alter flux through the Escherichia coli fatty acid synthesis by overexpressing acetyl-CoA carboxylase subunits and deleting cyclopropane fatty acid synthase (cfa) both failed to improve n-butanol tolerance in E. coli, indicating that additional components of the stress response are required to confer n-butanol resistance.Several promising routes for understanding both ferulic acid and n-butanol tolerance have been identified from L. brevis gene expression data. These insights may be used to guide further engineering of model industrial organisms to better tolerate both classes of inhibitors to enable facile production of biofuels from lignocellulosic biomass

Perspectives on the use of transcriptomics to advance biofuels

As a field within the energy research sector, bioenergy is continuously expanding. Although much has been achieved and the yields of both ethanol and butanol have been improved, many avenues of research to further increase these yields still remain. This review covers current research related with transcriptomics and the application of this high-throughput analytical tool to engineer both microbes and plants with the penultimate goal being better biofuel production and yields. The initial focus is given to the responses of fermentative microbes during the fermentative production of acids, such as butyric acid, and solvents, including ethanol and butanol. As plants offer the greatest natural renewable source of fermentable sugars within the form of lignocellulose, the second focus area is the transcriptional responses of microbes when exposed to plant hydrolysates and lignin-related compounds. This is of particular importance as the acid/base hydrolysis methods commonly employed to make the plant-based cellulose available for enzymatic hydrolysis to sugars also generates significant amounts of lignin-derivatives that are inhibitory to fermentative bacteria and microbes. The article then transitions to transcriptional analyses of lignin-degrading organisms, such as Phanerochaete chrysosporium, as an alternative to acid/base hydrolysis. The final portion of this article will discuss recent transcriptome analyses of plants and, in particular, the genes involved in lignin production. The rationale behind these studies is to eventually reduce the lignin content present within these plants and, consequently, the amount of inhibitors generated during the acid/base hydrolysis of the lignocelluloses. All four of these topics represent key areas where transcriptomic research is currently being conducted to identify microbial genes and their responses to products and inhibitors as well as those related with lignin degradation/formation.clos

Exopolysaccharide biosynthesis by a natural lactococcal ropy isolate

The genes coding for the expression of a ropy exopolysaccharide responsible for commercially desirable textural and rhealogical traits in fermented milk products by a natural lactococcal ropy isolate were sought. Using a transposon mutagenesis vector, pGh9:ISS1, three mutants lacking expression of the ropy exopolysaccharide were isolated. One of the mutants was chosen for further characterization. Using a Southern hybridization analysis, the interrupted gene was localized to the chromosome. The non-ropy mutant was further characterized and shown to be unable to produce ropy exopolysaccharide in fermented milk. A 2006 by fragment of the interrupted gene was sequenced. The DNA sequence over a short region showed homology to sugar transfer enzymes found in exopolysaccharide biosynthesis pathways. The DNA sequence was translated into its predicted amino acid sequences and two partial open reading frames of 236 and 338 amino acid residues in length were identified. These open reading frames were found to exhibit identity to glycosyltransferases present in exopolysaccharide biosynthesis pathways in other bacteria

Exopolysaccharide Expression in Lactococcus lactis subsp. cremoris Ropy352: Evidence for Novel Gene Organization

Lactococcus lactis subsp. cremoris Ropy352 produces two distinct heteropolysaccharides, phenotypically described as ropy and mucoid, when cultured in nonfat milk. One exopolysaccharide precipitated with 50% ethanol as a series of elongated threads and was composed of glucose and galactose in a molar ratio of 3:2. The second exopolysaccharide precipitated with 75% ethanol as a fine flocculant and consisted of galactose, glucose, and mannose with a molar ratio of 67:21:12. A mutant strain, L. lactis subsp. cremoris EK240, lacking the ropy phenotype did not produce the exopolysaccharide that precipitated with 50% ethanol; however, it produced the exopolysaccharide that precipitated with 75% ethanol, indicating that the former exopolysaccharide is essential for the ropy phenotype. Cultures of L. lactis subsp. cremoris Ropy352 in 10% nonfat milk reached a viscosity of 25 Pa-s after 24 h, while those of the nonropy L. lactis subsp. cremoris EK240 mutant did not change. A mutation abolishing ropy exopolysaccharide expression mapped to a region on a plasmid containing two open reading frames, epsM and epsN, encoding novel glycosyltransferases bordered by ISS1 elements oriented in the same direction. Sequencing of this plasmid revealed two other regions involved in exopolysaccharide expression, an operon located between partial IS981 and IS982 elements, and an independent gene, epsU. Two and possibly three of these regions are involved in L. lactis subsp. cremoris Ropy352 exopolysaccharide expression and are arranged in a novel fashion different from that of typical lactococcal exopolysaccharide loci, and this provides genetic evidence for exopolysaccharide gene reorganization and evolution in Lactococcus

An alternative biorefinery approach to address microalgal seasonality:Blending with spent coffee grounds

An effective method for the production of fuels and chemicals from microalgae is to ferment the carbohydrate fraction, extract the lipids and convert the resulting solids through hydrothermal liquefaction (HTL). In this process, known as Combined Algal Processing (CAP), multiple fuel precursors are produced effectively. However, one of the key challenges associated with a microalgae-based biorefinery is the reduced productivity of algae in the colder seasons. In this investigation, the potential for spent coffee grounds (SCG), a potentially valuable waste stream, to be blended with biomass from the microalgae Scenedesmus acutus (HCSD) to make up for the productivity shortfalls in periods of lower microalgae productivity to maximize the capacity for downstream equipment throughout the year was evaluated. Two different blend ratios were compared to only microalgae biomass or SCG, one representing winter season (40% microalgae and 60% SCG-blend 1) and another representing autumn and early spring (60% microalgae and 40% SCG-blend 2). Pretreatment of the blends showed higher monosaccharide release yields compared to microalgae alone, with an increase in mannose and galactose specifically. In the fermentation of the pretreated slurries, all the monosaccharides were consumed, resulting in ethanol titers of up to 23 g L-1 for the SCG blend, compared to 14 g L-1 ethanol for the algae alone. The lipid extraction from the blends resulted in yields of 95.5-99.7% (which translates to 173.8-193.5 kg per tonne of dry biomass processed in this biorefinery scenario) compared to 92.2% in HCSD (216.2 kg per tonne of dry biomass) and 68.1% in SCG (90.8 kg per tonne of dry biomass) alone. The residual solids left after fermentation and lipid extraction were converted via hydrothermal liquefaction (HTL) to produce bio-crude. The bio-crude yield was higher for microalgae (24.6%) than for the two blend cases (blend 1-17.5% and blend 2-19.7%). Theoretical energy calculations showed that the addition of SCG gave similar yields of fuel (gallon of gasoline equivalents) from the blends when compared to microalgae alone (94.7-96.5% depending on the blend of SCG). This work demonstrates that SCG can be easily incorporated with microalgae into a combined processing methodology and can therefore be used effectively during periods of lower availability of microalgae maintaining maximum operating levels of the conversion process equipment year-round. Moreover, co-processing algae with SCG not only leads to increased ethanol titers in the fermentation but also improves the lipid extraction yields. This journal is </p