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Ulva fenestrata is an economically and ecologically important green algal species with a large potential in seaweed aquaculture due to its high productivity, wide environmental tolerance, as well as interesting functional and nutritional properties. Here, we performed a series of manipulative cultivation experiments in order to investigate the effects of irradiance (50, 100, and 160 μmol photons m−2 s−1), temperature (13 and 18 °C), nitrate (pCO2 (200, 400, and 2500 ppm) on the relative growth rate and biochemical composition (fatty acid, protein, phenolic, ash, and biochar content) in indoor tank cultivation of Swedish U. fenestrata. High irradiance and low temperature were optimal for the growth of this northern hemisphere U. fenestrata strain, but addition of nutrients or changes in pCO2 levels were not necessary to increase growth. Low irradiance resulted in the highest fatty acid, protein, and phenolic content, while low temperature had a negative effect on the fatty acid content but a positive effect on the protein content. Addition of nutrients (especially nitrate) increased the fatty acid, protein, and phenolic content. High nitrate levels decreased the total ash content of the seaweeds. The char content of the seaweeds did not change in response to any of the manipulated factors, and the only significant effect of changes in pCO2 was a negative relationship with phenolic content. We conclude that the optimal cultivation conditions for Swedish U. fenestrata are dependent on the desired biomass traits (biomass yield or biochemical composition).

Seaweed biomass has the potential to become an important raw material for bio-based production. The aim of this study was to screen the overall composition of several seaweed species on the Swedish west coast, including some scarcely studied species, to provide fundamentals for evaluation of biorefining potential and to benchmark with already potentially industrially relevant species and commercially important land-based biomasses. Twenty-two common seaweed species (green, red, brown) were collected and the carbohydrate, ash, protein, water and metal contents were measured. Carbohydrate content varied between 237 and 557 g kg−1dry weight (dw), making it the largest constituent, on a dry weight basis, of most species in the study. Ash, which is considered unwanted in biorefining, ranged between 118 and 419 g kg−1dw and was the largest constituent in several seaweeds, which were therefore considered unsuitable for biorefining. Protein content was most abundant in the red seaweeds but was generally low in all species (59–201 g kg−1dw). High contents of several unwanted metals for processing or human consumption were found (e.g. aluminium, arsenic, copper, chromium and nickel), which need to be considered when utilizing seaweeds for certain applications. Potential targets for further biorefinery development mostly include species already known for their potential (Saccharina latissima,Laminaria digitataandChondrus crispus) while some, such asHalidrys siliquosaandDilsea carnosa, have not been previously noted. However, more detailed studies are required to explore biorefinery processes for these seaweeds, as well as how to potentially cultivate them.

In recent years, the interest in using seaweed for the sustainable production of commodities has been increasing as seaweeds contain many potentially worthwhile compounds. Thus, the extraction and refining processes of interesting compounds from seaweeds is a hot research topic but has been found to have problems with profitability for novel applications. To increase the economic potential of refining seaweed biomass, the content of the compounds of interest should be maximized, which can potentially be achieved through optimization of cultivation conditions. In this study, we studied how the monosaccharide composition of the green seaweed species Ulva fenestrata is influenced by the abiotic factors; irradiance, temperature, nitrate, phosphate, and pCO2. It was evident that lower nitrate concentration and cultivation at elevated temperature increased monosaccharide contents. A 70% increase in iduronic acid and a 26% increase in rhamnose content were seen under elevated irradiance and temperature conditions, though the absolute differences in monosaccharide concentration were small. Irradiance and nitrate impacted the ratio between iduronic and rhamnose, which is an indicator of the ulvan structure. These results could potentially be utilized to coax the ulvan towards specific bioactivities, and thus have a considerable impact on a potential biorefinery centered around Ulva.

Structural, functional and nutritional properties of protein recovered from brown seaweed, S. latissima with alkaline solubilization/isoelectric precipitation as a function of different post-harvest stabilization methods were studied. The latter included freezing at −20 °C/-80 °C, oven-drying, sun-drying, freeze-drying and ensilaging. Also, the efficacy of freeze/thaw-aided precipitation (F/T) in improving protein recovery of the process was evaluated. The freeze-dried, oven-dried, and −20 °C frozen seaweeds resulted in significantly higher protein yield than the −80°C-frozen, sun-dried and ensiled biomasses. F/T increased protein precipitation and doubled total protein yield. Sun-drying and −20°C-freezing caused extensive protein degradation as revealed by SDS-PAGE and HP-SEC, while oven-drying altered the seaweed protein structure with less α-helices. Functional properties of the seaweed proteins were remarkably affected by stabilization condition and F/T, but nutritional value of the proteins was only dependent on stabilization method. Thus, to efficiently recover seaweed proteins, its post-harvest stabilization condition must be carefully chosen based on the final application of the proteins.

Globally, there is a need for novel vegetarian protein sources. We recently showed that the pH-shift process, using alkaline protein solubilization followed by isoelectric precipitation, is an efficient way to produce extracts with high protein concentrations from Ulva lactuca (>50% on a dry matter basis). However, the total protein yield was low, and to improve this, the effects of adding ulvan lyase, preincubating the seaweed homogenate at pH 8.5 and using different protein extraction temperatures (8 °C, RT and 40 °C), were evaluated in this study. Addition of ulvan lyase reduced protein solubility but increased the precipitation. Incubation at pH 8.5, without ulvan lyase added, significantly increased both protein solubility and precipitation at 8 °C and RT. Temperature per se had no effect on protein solubility, while protein precipitation increased with decreasing temperature. Highest protein yield (29%) was achieved when keeping the process at 8 °C with a preincubation step at pH 8.5 for 1 h. By these process modifications, the yield was 3.2 times higher than achieved by the control process (9.2%).

Members of green macroalgae cause green tides in eutrophicated coastal waters. These green tides pose an environmental issue and an economic burden on coastal municipalities. The biomass from these green tides has a potential to be used as feedstock in biorefinery due to the content of interesting biomacromolecules. Ulvan, an anionic water-soluble polysaccharide, is one of such components, and its depolymerisation to high-value oligosaccharides or fermentable monosaccharides would bring value to green tide biomass which is otherwise left to decompose. However, only a few ulvan depolymerising enzymes are studied to date. Ulvan lyases depolymerise ulvan, via the β-elimination mechanism, leading to release of oligosaccharides with an unsaturated 4‑deoxy‑l‑threo‑hex‑4‑enopyranosiduronic acid at the non-reducing end. In this study, we have identified the presence of two different domains, a catalytic and a non-catalytic, in a putative ulvan lyase from Formosa agariphila KMM 3901. We overexpressed, purified, and biochemically characterised the full-length ulvan lyase, which was found to be most active at a temperature of 45 °C and pH 8.5. It exhibited high specificity for ulvan and did not degrade heparan sulphate, chondroitin sulphate, alginate, pectin or xanthan. Detailed analyses of end products of the enzymatic degradation of ulvan using 1H NMR and LC-MS revealed a disaccharide with an unsaturated uronic acid (∆) linked to 3‑sulphated rhamnose (Rha3S), trisaccharide with xylose (Xyl) flanked by Rha3S (Rha3S-Xyl-Rha3S), tetrasaccharide with an unsaturated uronic acid at the non-reducing end (∆Rha3S-Xyl-Rha3S) and pentasaccharides (Rha3S-Xyl-Rha3S-Xyl-Rha3S and branched ∆Rha3S-Xyl-(∆)Rha3S) as the principal end products. We also found that the catalytic domain that lacks the non-catalytic carbohydrate binding module exhibited higher affinity for the soluble ulvan and efficiently depolymerised it. This study reveals the characteristics of the endolytic ulvan lyase, which is a member of the ulvan utilisation loci in Formosa, and points towards the potential ulvan depolymerisation applications in Ulva biorefinery.

Hydrothermal liquefaction (HTL) of macroalgae offers a promising route to advanced biofuel production, although the distinct biochemical compositions of different macroalgae species can lead to widely different product yields and compositions. On the basis of this, there is an implicit assumption that there exists a universal optimal feedstock species for a bioenergy-based biorefinery, which could be exploited across a wide region. However, no studies to date have examined the effect of this large geographical variation on a single macroalgae species for biofuel production. In this study, 24 samples of Ulva intestinalis were collected along 1200 km of Swedish coastline and assessed as a feedstock for HTL. Significant variation incomposition was observed between samples from Baltic and Atlantic regions, but substantial variation also existed between sites within close proximity. This was reflected in the HTL biocrude oil yields, which varied between 9 and 20% (14−28% dry and ash-free basis) across the sample set. In a number of cases, greater variation was seen for adjacent sites than for sites at oppositeends of the sampling spectrum. Biocrude oil yields in this study also differed substantially from those previously obtained for U.intestinalis from the United Kingdom and Vietnam. Localized environmental conditions affected the HTL product composition significantly, in particular, the elemental distribution within the sample set. The variability observed in this study suggests that no single species will be dominant within a macroalgal biorefinery concept, but rather a species would need to be selected tomatch the needs of the exact local environment

Macroalgal biomass has the potential to become an important source of chemicals and commodities in a future biorefinery. Currently, production of macroalgal biomass is expensive and the content of high-value compounds is often low. Therefore, in this study the biochemical composition of Ulva intestinalis along the Swedish west coast and the east coast up to Stockholm was assessed with the aim of determining how the content of potentially valuable compounds, such as rhamnose, iduronic acid and PUFAs, could be maximized by utilizing natural variation in the choice of marine cultivation site. Along the investigated coastline, the salinity dropped from 19.4‰ at high latitudes along the west coast to 5.4‰ at Stockholm. Nitrogen and phosphorus availability varied, while temperature was similar at all locations. The two major components of biomass, carbohydrates and ash, varied inversely with the highest content of ash in the west and carbohydrates in the east. In addition, total fatty acids were significantly higher in west coast samples at 3.2 g 100 g–1 dw, with a higher proportion of mono- and polyunsaturated fatty acids. Some health-beneficial fatty acids were found, including EPA and DPA, at 10–50 mg 100 g–1 dw, respectively. The metal content and elemental composition varied widely, probably due to the influence of specific local conditions. The P content was correlated with the phosphorus concentration in waters at the locations. In PCA analysis, the monosaccharides constituting the cell wall polysaccharide ulvan were found to vary by geographical location, with higher levels possibly associated with lower salinities. However, only glucuronic acid differed significantly between sites. These results show the considerable geographical variability in the composition of Swedish U. intestinalis and suggest that different salinities could be used to create a lipid- or carbohydrate-rich biomass.

Seaweed cultivation and processing industries could contribute to sustainable blue growth and the European bioeconomy. This article contributes a case study evaluation of environmental sustainability of preserved brown seaweed Saccharina latissima by means of environmental life cycle assessment of a pilot facility in Sweden. The study accounts for nutrient bioremediation and carbon capture and includes two alternative hatchery processes, a 2-ha longline cultivation, and four alternative preservation methods (hang-drying outdoors, heated air-cabinet drying, ensiling, and freezing). The study found that as a result of carbon capture and nitrogen and phosphorus uptake (bioremediation) by seaweed, more CO2 and PO4 equivalents are (temporarily) absorbed than emitted by the supply chain. The extent of emissions is most affected by preservation methods undertaken. Impact profiles of the supply chain show that the greatest impact shares result from freezing and air-cabinet drying, both the two most energy-intensive processes, followed by the cultivation infrastructure, highlighting strategic optimization opportunities. Hatchery processes, harvesting, and the low-energy ensilage and hang-drying outdoors were found to have relatively small impact shares. These findings presage the environmentally friendliness of seaweed-based products by documenting their potential to mitigate eutrophication and climate change, even when taking a life cycle perspective.

Microalgal biomass represents a potential third generation feedstock that could be utilised as a source of carbohydrates for fermentative production of a range of platform biochemicals. Identifying microalgal strains with high biomass and carbohydrate productivities while also being amenable to downstream processes is key in improving the feasibility of these processes. Utilising marine microalgae capable of growing in seawater will decrease reliance on freshwater resources and improve the sustainability of production. This study screened several marine microalgae believed to accumulate carbohydrates to find new high performing strains. Four strains had high growth rates and accumulated carbohydrates > 35% DW under stress. The strain Chlorella salina demonstrated the highest biomass and carbohydrate productivity, and alkaline autoflocculation (4 mM NaOH) enabled biomass recoveries > 95% efficiency, resulting in an 8–10 × concentration of the culture. Under nutrient replete conditions, biomass productivity reached 0.6 g L −1 d −1 , significantly greater than that of nitrogen starved cultures. However, nitrogen starvation rapidly increased carbohydrate content to > 50% DW in 2 days, resulting in carbohydrate productivities > 0.20 g L −1 d −1 . Chlorella salina partitions the products of photosynthesis preferentially into carbohydrate synthesis under nitrogen starvation. A greater understanding of cellular physiology and carbon partitioning in response to nutrient stress will enable better control and optimisation of the bio-processes. This study has identified a potentially high performance marine microalga for carbohydrate production that is also amenable to low-cost harvesting.

Saccharina latissima biomass cultivated along the Swedish west coast was subjected to four different scalable preservation methods after harvest; freezing, sun-drying, oven-drying and ensiling. Freeze-drying and freezing at −80 °C were also included to provide dry and wet references. The effects of the different preservation methods on the composition of Saccharina biomass (on dry weight, DW, basis), and the recovery as well as properties of high-quality protein, alginate and biogas were evaluated. Sun-drying significantly reduced protein, alginate and fatty acid content of the seaweeds and thereby concentrated ash in the biomass compared to the other methods. Protein/amino acids and fatty acids were significantly concentrated in ensiled biomass, while mannitol and laminarin were reduced compared to the other biomasses. Oven-drying and −20 °C freezing affected the composition the least, with lower ash content and alterations in some specific amino and fatty acids. Sun-drying and ensiling resulted in significantly lower protein solubility at high pH compared to the other biomasses which translated into the lowest total seaweed protein recovery using the pH-shift process. Highest protein yield was obtained with the freeze-dried reference. Ensiling lead to a significant decrease in the molecular weight of alginate, while sun-drying caused a negative effect on alginate by inducing a shift in the guluronic and mannuronic acids composition of alginate. Sun-drying gave the lowest methane yield in the anaerobic digestion experiments while freezing at −80 °C gave the highest yield, closely followed by freezing at −20 °C and ensiling. To conclude, preservation methods must be carefully chosen to protect the valuable component in Saccharina latissima, and to achieve an efficient downstream processing ultimately yielding high quality products as part of a seaweed biorefinery.

The high ash content and varying ash composition in algal biomass is often mentioned as problematic if to be used for thermal energy conversion. This paper suggests an approach where detailed information on ash composition and predicted ash formation reactions are basis for successful remedies enabling the use of fuels considered to be difficult. The procedure is demonstrated on seaweed (Saccharina latissima) cultivated for biorefinery purposes. The ash composition of the seaweed was found suitable for co-combustion with Miscanthus x giganteus, an energy crop high in alkali and silicon. Fuel mixtures were combusted in a bubbling fluidized bed reactor and ash samples were analyzed by SEM-EDS and XRD. The results showed that Ca from the seaweed was very reactive and thus efficient in solving the silicate melting problems. The fuel design approach was proven successful and the potential for using otherwise difficult seaweed fuels in synergetic co-combustion was demonstrated. (C) 2017 Elsevier B.V. All rights reserved.

This study monitored the biomass composition of Saccharina latissima during a potential harvesting season on the West coast of Sweden, in order to find suitable harvest times for biorefinery purposes. Specimens of S. latissima were sampled at three locations in June, August and October and the biomass was analysed for its macromolecular composition, as well as for the content of several specific compounds, e.g. sugars and fatty acids. PERMANOVA analyses showed that there was a significant difference in the biomass composition among time points. The total carbohydrate concentration was lowest in June and peaked at 360 mg g-1 dry weight in August, while the mannitol content was highest, 90 mg g-1, in June and decreased throughout the sampling period. Total protein and fatty acid concentrations were found to be approximately 80 and 3 mg g-1, respectively, with relatively little variation over time. Overall, there was little spatial variation in the macromolecular composition, although the concentration of some specific monosaccharides and fatty acids, as well as the total phenolic content, differed among localities. We discuss the implications of the observed variation in biomass composition of S. latissima for future biorefinery purposes.

The cell wall polysaccharide ulvan was isolated from two species of the seaweed Ulva collected along the Swedish west coast. Acidic extraction was benchmarked against hot water extraction with enzymatic purification and against commercial ulvan. Extracted ulvan contained 11–18 % g/g of ash, some protein (up to 1.3 % g N/g) but minimal colored impurities. The ulvans had high molecular weights (660,000–760,000 g/mol) and were composed of 77–79 % g/g carbohydrates, mainly rhamnose, xylose, glucose, glucuronic acid, and iduronic acid. The extraction protocol and the ulvan source strongly impact the molecular weight and the chemical composition. Acidic extraction caused almost complete desulfation of the isolated ulvan while the other method preserved a significant degree of SO3 substituents. Elemental analysis of ash remaining after thermal degradation showed presence of common mineral elements such as Na, Ca, Mg, Al, and K, but none of the heavy metals Pb, Hg, or As.

Harvesting microalgal cultures is often energetically intensive and costly. To improve efficiencies, a two-step harvesting method utilising alkaline flocculation and sedimentation to pre-concentrate cultures can be used prior to centrifugation. When applied to the microalga Chromochloris zofingiensis, high rates of sedimentation (>90%) were found at low concentrations of base (

Ulvan, which is one of the major structural polysaccharides of the cell walls of green macroalgae, is degraded by ulvan lyases via the β-elimination mechanism with the release of oligosaccharides that have unsaturated 4-deoxy-L-threo-hex-4-enopyranosiduronic acid (∆) at the non-reducing end. These ulvan lyases belong to the PL24 or PL25 or PL28 family in the CAZy database. In this study, we identify and biochemically characterise a periplasmic novel broad-spectrum ulvan lyase from Formosa agariphila KMM 3901. The lyase was overexpressed in Escherichia coli, and the purified recombinant enzyme depolymerised ulvan in an endolytic manner with a Km of 0.77 mg/ml, and displayed optimum activity at 40 °C and pH 8. This lyase also degraded heparan sulphate and chondroitin sulphate. Detailed analyses of the end-products of the enzymatic degradation of ulvan using 1H- and 13C-NMR and LC-MS revealed an unsaturated disaccharide (∆Rha3S) and a tetrasaccharide (∆Rha3S-Xyl-Rha) as the principal end-products. In contrast to the previously described ulvan lyases, this novel lyase is mostly composed of α-helices that form an (α/α)6 incomplete toroid domain and displays a remarkably broad-spectrum activity. This novel lyase is the first member of a new family of ulvan lyases.

Fractionation of plant and animal raw materials by pH-shift processing has been widely applied to purify proteins. The principle is to solubilize proteins at high or low pH, removing debris and precipitating the proteins near their isoelectric point. We here describe the pH-shift process on commercially available Nannochloropsis oculata. The partitioning of major nutrients into the various fractions of the process was studied. Proteins were found to exhibit maximal solubility between pH7 and 10, with a minimal solubility below pH4. Two process versions were investigated in this study, with solubilization at either pH7 (native pH) or 10; both versions were precipitated at pH3. Up to 85% of both the protein and total fatty acids were recovered in the final product, compared to the initial algal slurry. Protein, total fatty acids and carbohydrates were concentrated in the final product, while the ash content was lower compared to the starting material. From a processing point of view, solubilization of Nannochloropsis at native pH was found to be preferable, since less chemicals were consumed compared to high-pH solubilization. Owing to its content of protein and (total) fatty acids (23 and 12% of dry weight, respectively), the latter enriched in eicosapentaenoic acid (EPA), the product of the pH-shift process has potential as a functional food ingredient.

Microalgal biotechnology has yielded a range of products for different consumer markets, but large scale production for bulk commodities is limited by the cost and environmental impact of production. Nutrient requirements for large-scale production contribute significantly to the cost and environmental impact of microalgal biomass production and should subsequently be addressed by more careful sourcing of nutrients. This study assessed the use of nitrogen and phosphorus contained in effluents from anaerobic digestion of food waste to cultivate the marine microalga Nannochloropsis sp. With suitable dilution, effluent could replace 100% of nitrogen demands and 16% of required phosphorus, without significant impacts on growth or biomass productivity. Additional phosphorus requirements could be decreased by increasing the N:P molar ratio of the media from 16:1 to 32:1. Nannochloropsis sp. accumulated lipid up to 50% of dry weight under N-stress, with significant increases in the content of saturated and mono-unsaturated fatty acids. Using empirical data generated in this study, the cost and environmental impact of nitrogen and phosphorus supply was assessed versus the use of fertilizers for biomass and biodiesel production. Nutrient requirements predicted by the Redfield Ratio overestimating impacts by as much as 140% compared to empirical data. By utilising residual nutrients and optimising nutrient supply, the cost and environmental impact of nitrogen and phosphorus were decreased by >90% versus the use of artificial fertilizers. This study demonstrates the importance of using empirical data for process evaluation and how anaerobic digestate effluent derived nutrients can contribute to the sustainability of algal biomass production.

The microalga Porphyridium cruentum (Rhodophyta) has several industrial and pharmaceutical uses, especially for its polysaccharide production. This study aimed to investigate the influence of nitrogen levels as reflected by altered N:P ratios on the production and content of biomass and carbohydrate. N:P molar ratios were altered in batch cultures to range from 1.6 to 50 using the Redfield ratio of 1:16 as reference. Algal growth (estimated as final cell number, biomass concentration and maximum specific growth rate) was negatively affected at low N:P ratios. The optimal N:P ratio for growth was identified at 35–50, with specific growth rates of 0.19 day−1 and maximum cell concentrations of 59·108 cells L−1 and 1.2 g dry weight of biomass L−1. In addition, variation in cell size was seen. Cells with larger diameters were at higher N:P ratios and smaller cells at lower ratios. The cellular carbohydrate content increased under reduced nitrogen availability. However, because accumulation was moderate at the lowest N:P ratio, 0.4 g per g dry weight biomass compared to 0.24 at the Redfield ratio of 16:1, conditions for increased total carbohydrate formation were identified at the N:P ratios optimal for growth. Additionally, carbohydrates were largely accumulated in late exponential to stationary phase.

The pH-shift process fractionates biomass into soluble proteins and insoluble fractions, followed by precipitation and recovery of the solubilized proteins. Nannochloropsis oculata in seawater was subjected to the pH-shift process, followed by digestion of various intermediates and product fractions of the process, using the Infogest in vitro digestion model (Minekus et al., 2014) with added gastric lipase. As measures for protein and lipid accessibility, degrees of protein hydrolysis and fatty acid liberation were assessed post-digestion and compared to the amounts of peptide bonds and total fatty acids present in the raw materials. Results showed that neither proteins nor lipids of intact Nannochloropsis cells were accessible to the mammalian digestive enzymes used in the digestion model. Cell disruption, and to a lesser extent, further pH-shift processing with protein solubilisation at pH 7 or pH 10, increased the accessibility of lipids. For proteins, differences amongst the pH-shift processed materials were non-significant, though pre-freezing the product prior to digestion increased the accessibility from 32% to 47%. For fatty acids, pH-shift process-products gave rise to 43% to 52% lipolysis, with higher lipolysis for products solubilised at pH 10 as opposed to pH 7. Our results indicate the importance of processing to produce an algal product that has beneficial nutritional properties when applied as food or feed.

The fermentation performance of Saccharomyces cerevisiae in the cellulose to ethanol conversion process is largely influenced by the components of pretreated biomass. The insoluble solids in pretreated biomass predominantly constitute cellulose, lignin, and -to a lesser extent- hemicellulose. It is important to understand the effects of water-insoluble solids (WIS) on yeast cell physiology and metabolism for the overall process optimization. In the presence of synthetic lignocellulosic inhibitors, we observed a reduced lag phase and enhanced volumetric ethanol productivity by S. cerevisiae CEN.PK 113-7D when the minimal medium was supplemented with WIS of pretreated birch or spruce and glucose as the carbon source. To investigate the underlying molecular reasons for the effects of WIS, we studied the response of WIS at the proteome level in yeast cells in the presence of acetic acid as an inhibitor. Comparisons were made with cells grown in the presence of acetic acid but without WIS in the medium. Altogether, 729 proteins were detected and quantified, of which 246 proteins were significantly up-regulated and 274 proteins were significantly down-regulated with a fold change≥1.2 in the presence of WIS compared to absence of WIS. The cells in the presence of WIS up-regulated several proteins related to cell wall, glycolysis, electron transport chain, oxidative stress response, oxygen and radical detoxification and unfolded protein response; and down-regulated most proteins related to biosynthetic pathways including amino acid, purine, isoprenoid biosynthesis, aminoacyl-tRNA synthetases and pentose phosphate pathway. Overall, the identified differentially regulated proteins may indicate that the likelihood of increased ATP generation in the presence of WIS was used to defend against acetic acid stress at the expense of reduced biomass formation. Although, comparative proteomics of cells with and without WIS in the acetic acid containing medium revealed numerous changes, a direct effect of WIS on cellular physiology remains to be investigated.

A bioanalytical method for indirect determination of eflornithine enantiomers in 75 microL human plasma has been developed and validated. L- and D-eflornithine were derivatized with o-phthalaldehyde and N-acetyl-L-cysteine to generate diastereomers which were separated on two serially connected Chromolith Performance columns (RP-18e 100 x 4.6 mm i.d.) by a isocratic flow followed by a gradient flow for elution of endogenous compounds. The diastereomers were detected with UV (340 nm). The between-day precisions for L- and D-eflornithine in plasma were 8.4 and 2.3% at 3 microm, 4.0 and 5.1% at 400 microm, and 2.0 and 3.7% at 1000 microm. The lower limit of quantification was determined to be 1.5 microm, at which precision was 14.9 and 9.9% for L- and D-eflornithine, respectively.

The methionine salvage pathway, also called the 5'-methylthioadenosine (MTA) cycle, recycles the sulfur of MTA, which is a by-product in the biosyntheses of polyamine and the plant hormone ethylene. MTA is first converted to 5'-methylthioribose-1-phosphate either by MTA phosphorylase or the combined action of MTA nucleosidase and 5'-methylthioribose kinase. Subsequently, five additional enzymatic steps, catalyzed by four or five proteins, will form 4-methylthio-2-oxobutyrate, the deaminated form of methionine. The final transamination is achieved by transaminases active in the amino acid biosynthesis. This pathway is present with some variations in all types of organisms and seems to be designed for a quick removal of MTA achieved by high affinities of the first enzymes. During evolution some enzymes have attained additional functions, like a proposed role in nuclear mRNA processing by the aci-reductone dioxygenase. For others the function seems to be lost due to conditions in specific ecological niches, such as, presence of sulfur and/or absence of oxygen resulting in that, for example, Escherichia coli is lacking a functional pathway. The pathway is regulated as response to sulfur availability and take part in the regulation of polyamine synthesis. Some of the enzymes in the pathway show separate specificities in different organisms and some others are unique for groups of bacteria and parasites. Thus, promising targets for antimicrobial agents have been identified. Other medical topics to which this pathway has connections are cancer, apoptosis, and inflammatory response.

In general, it is believed that fermentation by yeast under harsh industrial conditions, especially if substrates such as wood hydrolysate or lignocellulosic substrates are used, requires the use of so-called industrial strains. In order to check whether this is always true, a comparison of performance was made using two industrial strains and four commonly used laboratory strains, the haploid and diploid versions of CEN-PK and X2180, under industrially relevant stress conditions. The industrial strains were a Swedish commercial baker's yeast strain and a strain previously isolated from an industrial bioethanol production plant using lignocellulosic substrate. Stress conditions included, apart from growth in the lignocellulosic substrate itself, elevated concentrations of glucose, NaCl, ethanol, and lactate as well as low pH. Results showed that, indeed, the strain adapted to lignocellulosic substrate also possessed the highest growth rate as well as shortest duration of the lag phase in this type of medium. However, the higher the additional stress level, the lower the difference compared to other strains, and X2180 in particular displayed a high resistance to these additional stress conditions. Furthermore, no difference in performance could be detected between the haploid or diploid versions of the laboratory strains. It might be that, at least under some circumstances, a laboratory strain such as X2180 could be an industrially attractive production organism with the advantage of facilitating the possibilities for making controlled genetic manipulations.

The non-ethylene producing yeast, Saccharomyces cerevisiae, was transformed into an ethylene producer by introducing the ethylene forming enzyme from the plant pathogenic bacterium Pseudomonas syringae. Cultivation of the metabolically engineered strain was performed in well-controlled bioreactors as aerobic batch cultures with an on-line monitoring of ethylene production. The highest productivity was obtained during the respiro-fermentative growth on glucose but there was also a significant rate of formation during the subsequent phase of ethanol respiration. Furthermore, investigations were performed whether substitution of the original nitrogen source, NH(4)(+), for glutamate could improve productivity and yield of ethylene even more. The rationale being that one of the substrates for the enzyme is 2-oxoglutarate and this compound can be formed from glutamate in a single reaction. Indeed, there was a substantial improvement in the rate of production and the final yield of ethylene was almost three times higher when NH(4)(+) was replaced by glutamate.

Microalgae have great prospects as a sustainable resource of lipids for refinement into nutraceuticals and biodiesel, which increases the need for detailed insights into their intracellular lipid synthesis/storage mechanisms. As an alternative strategy to solvent- and label-based lipid quantification techniques, we introduce time-gated coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring lipid contents in living algae, despite strong autofluorescence from the chloroplasts, at approximately picogram and subcellular levels by probing inherent molecular vibrations. Intracellular lipid droplet synthesis was followed in Phaeodactylum tricornutum algae grown under (1) light/nutrient-replete (control [Ctrl]), (2) light-limited (LL), and (3) nitrogen-starved (NS) conditions. Good correlation (r 2 = 0.924) was found between lipid volume data yielded by CARS microscopy and total fatty acid content obtained from gas chromatography-mass spectrometry analysis. In Ctrl and LL cells, micron-sized lipid droplets were found to increase in number throughout the growth phases, particularly in the stationary phase. During more excessive lipid accumulation, as observed in NS cells, promising commercial harvest as biofuels and nutritional lipids, several micron-sized droplets were present already initially during cultivation, which then fused into a single giant droplet toward stationary phase alongside with new droplets emerging. CARS microspectroscopy further indicated lower lipid fluidity in NS cells than in Ctrl and LL cells, potentially due to higher fatty acid saturation. This agreed with the fatty acid profiles gathered by gas chromatography-mass spectrometry. CARS microscopy could thus provide quantitative and semiqualitative data at the single-cell level along with important insights into lipid-accumulating mechanisms, here revealing two different modes for normal and excessive lipid accumulation.

Recent studies of Saccharomyces cerevisiae revealed sensors that detect extracellular amino acids (Ssy1p) or glucose (Snf3p and Rgt2p) and are evolutionarily related to the transporters of these nutrients. An intriguing question is whether the evolutionary transformation of transporters into nontransporting sensors reflects a homeostatic capability of transporter-like sensors that could not be easily attained by other types of sensors. We previously found SSY1 mutants with an increased basal level of signaling and increased apparent affinity to sensed extracellular amino acids. On this basis, we propose and test a general model for transporter-like sensors in which occupation of a single, central ligand binding site increases the activation energy needed for the conformational shift between an outward-facing, signaling conformation and an inward-facing, nonsignaling conformation. As predicted, intracellular leucine accumulation competitively inhibits sensing of extracellular amino acids. Thus, a single sensor allows the cell to respond to changes in nutrient availability through detection of the relative concentrations of intra- and extracellular ligand.

Recent studies of Saccharomyces cerevisiae revealed sensors that detect extracellular amino acids (Ssy1p) or glucose (Snf3p and Rgt2p) and are evolutionarily related to the transporters of these nutrients. An intriguing question is whether the evolutionary transformation of transporters into nontransporting sensors reflects a homeostatic capability of transporter-like sensors that could not be easily attained by other types of sensors. We previously found SSY1 mutants with an increased basal level of signaling and increased apparent affinity to sensed extracellular amino acids. On this basis, we propose and test a general model for transporter- like sensors in which occupation of a single, central ligand binding site increases the activation energy needed for the conformational shift between an outward-facing, signaling conformation and an inward-facing, nonsignaling conformation. As predicted, intracellular leucine accumulation competitively inhibits sensing of extracellular amino acids. Thus, a single sensor allows the cell to respond to changes in nutrient availability through detection of the relative concentrations of intra- and extracellular ligand.

Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 μmol/g, while nitrogen-starved cells still contained approximately 6 μmol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period.

Texto integral disponível através do editor Two genes YER081W and YIL074C, renamed SER3 and SER33, respectively, which encode phosphoglycerate dehydrogenases in Saccharomyces cerevisiae were identified. These dehydrogenases catalyze the first reaction of serine and glycine biosynthesis from the glycolytic metabolite 3-phosphoglycerate. Unlike either single mutant, the ser3Delta ser33Delta double mutant lacks detectable phosphoglycerate dehydrogenase activity and is auxotrophic for serine or glycine for growth on glucose media. However, the requirement for the SER-dependent "phosphoglycerate pathway" is conditional since the "glyoxylate" route of serine/glycine biosynthesis is glucose-repressed. Thus, in cells grown on ethanol both expression and activity of all SER-encoded proteins are low, including the remaining enzymes of the phosphoglycerate pathway, Ser1p and Ser2p. Moreover the available nitrogen source regulates the expression of SER genes. However, for only SER33, and not SER3, expression was regulated in relation to the available nitrogen source in a coordinated fashion with SER1 and SER2. Based on these mRNA data together with data on enzyme activities, Ser33p is likely to be the main isoenzyme of the phosphoglycerate pathway during growth on glucose. Moreover, since phosphoglycerate dehydrogenase activity requires NAD(+) as cofactor, deletion of SER3 and SER33 markedly affected redox metabolism as shown by substrate and product analysis.

Product concentrations may be estimated from gas analysis or calorimetric measurements. These techniques of analysis are fast, simple, and provide a continuous output. To check the accuracy of these estimations, the anaerobic ethanol formation of an industrial strain of the yeast Saccharomyces cerevisiae was selected as a test system. Cultivations were carried out as batch cultures with different nitrogen sources (ammonium, glutamate and a mixture of amino acids) and 20 g/l of glucose as the carbon and energy source. The results showed that there was a good agreement between measured and calculated ethanol values. However, especially when using heat data the results are very sensitive to small changes in the stoichiometry of the catabolic reaction, i.e. ethanol is not the only product but there are also minor amounts of glycerol and acetate. The problems generated by these products when using heat data to calculate ethanol concentrations are discussed in detail.

The response of glycolysis to exposure of glucose in non-growing S. cerevisiae cells from diauxic shift was monitored. The result was compared to a kinetic model of glycolysis with branches to glycogen, trehalose, glycerol, and succinate. Experimental data at steady-state concentrations of metabolites in the upper part agreed well between the model and experiments. Larger discrepancies were found in the lower part of glycolysis and as concerns the rates of glycerol and ethanol formation. Further improvements of the model are needed and new branches of glycolysis have to be identified and included. The control of the ethanol flux were exerted by the uptake since the flux control coefficient for glucose transport was 1.09.

To prevent the loss of raw material in ethanol production by anaerobic yeast cultures, glycerol formation has to be reduced. In theory, this may be done by providing the yeast with amino acids, since the de novo cell synthesis of amino acids from glucose and ammonia gives rise to a surplus of NADH, which has to be reoxidized by the formation of glycerol. An industrial strain of Saccharomyces cerevisiae was cultivated in batch cultures with different nitrogen sources, i.e., ammonium salt, glutamic acid, and a mixture of amino acids, with 20 g of glucose per liter as the carbon and energy source. The effects of the nitrogen source on metabolite formation, growth, and cell composition were measured. The glycerol yields obtained with glutamic acid (0.17 mol/mol of glucose) or with the mixture of amino acids (0.10 mol/mol) as a nitrogen source were clearly lower than those for ammonium-grown cultures (0.21 mol/mol). In addition, the ethanol yield increased for growth on both glutamic acid (by 9%) and the mixture of amino acids (by 14%). Glutamic acid has a large influence on the formation of products; the production of, for example, alpha-ketoglutaric acid, succinic acid, and acetic acid, increased compared with their production with the other nitrogen sources. Cultures grown on amino acids have a higher specific growth rate (0.52 h-1) than cultures of both ammonium-grown (0.45 h-1) and glutamic acid-grown (0.33 h-1) cells. Although the product yields differed, similar compositions of the cells were attained. The NADH produced in the amino acid, RNA, and extracellular metabolite syntheses was calculated together with the corresponding glycerol formation. The lower-range values of the theoretically calculated yields of glycerol were in good agreement with the experimental yields, which may indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential.

Background While simultaneous saccharification and co-fermentation (SSCF) is considered to be a promising process for bioconversion of lignocellulosic materials to ethanol, there are still relatively little demo-plant data and operating experiences reported in the literature. In the current work, we designed a SSCF process and scaled up from lab to demo scale reaching 4% (w/v) ethanol using xylose rich corncobs. Results Seven different recombinant xylose utilizing Saccharomyces cerevisiae strains were evaluated for their fermentation performance in hydrolysates of steam pretreated corncobs. Two strains, RHD-15 and KE6-12 with highest ethanol yield and lowest xylitol yield, respectively were further screened in SSCF using the whole slurry from pretreatment. Similar ethanol yields were reached with both strains, however, KE6-12 was chosen as the preferred strain since it produced 26% lower xylitol from consumed xylose compared to RHD-15. Model SSCF experiments with glucose or hydrolysate feed in combination with prefermentation resulted in 79% of xylose consumption and more than 75% of the theoretical ethanol yield on available glucose and xylose in lab and PDU scales. The results suggest that for an efficient xylose conversion to ethanol controlled release of glucose from enzymatic hydrolysis and low levels of glucose concentration must be maintained throughout the SSCF. Fed-batch SSCF in PDU with addition of enzymes at three different time points facilitated controlled release of glucose and hence co-consumption of glucose and xylose was observed yielding 76% of the theoretical ethanol yield on available glucose and xylose at 7.9% water insoluble solids (WIS). With a fed-batch SSCF in combination with prefermentation and a feed of substrate and enzymes 47 and 40 g l-1 of ethanol corresponding to 68% and 58% of the theoretical ethanol yield on available glucose and xylose were produced at 10.5% WIS in PDU and demo scale, respectively. The strain KE6-12 was able to completely consume xylose within 76 h during the fermentation of hydrolysate in a 10 m3 demo scale bioreactor. Conclusions The potential of SSCF is improved in combination with prefermentation and a feed of substrate and enzymes. It was possible to successfully reproduce the fed-batch SSCF at demo scale producing 4% (w/v) ethanol which is the minimum economical requirement for efficient lignocellulosic bioethanol production process.