PURPOSE:The purpose of this work was to determine the pro-and anti-inflammatory properties of the single-cell organism Euglena gracilis (EG) and various fractions of its whole biomass. METHODS:Heterotrophically grown EG was tested, along with its aqueous fraction (E-AQ), the intact linear β-glucan paramylon granules (PAR), and alkaline-solubilized paramylon. Peripheral blood mononuclear cell cultures were treated with the test products and analyzed for a variety of cellular responses. Immune cell activation was evaluated by flow cytometry detection of CD69 levels on CD3-CD56+ NK cells, CD3+CD56+ NKT cells, and monocytes, and cytokines were analyzed from the cell culture supernatants. Antioxidant capacity was measured by Folin-Ciocalteu assay and cellular antioxidant protection and MTT assays. RESULTS:EG and E-AQ were the most effective in driving immune cell responses as measured by CD69 upregulation on NK and NKT cells and proinflammatory (tumor necrosis factor, IL-6, IL-1β) cytokine production. None of the test products effectively stimulated monocyte. EG and PAR inhibited reactive oxygen species under conditions of oxidative stress. E-AQ contained antioxidants capable of providing cellular antioxidant protection from oxidative damage and protection of mitochondrial function under inflammatory conditions. CONCLUSION:The effects of EG on immune function are only partially attributable to the content of the β-glucan, paramylon. The regulation of additional cellular responses, such a reactive oxygen species production and resistance to oxidative stress, is likely mediated by currently unknown molecules found in the EG cell.

Algal biofuels have the potential to curb the emissions of greenhouse gases from fossil fuels, but current growing methods fail to produce fuels that meet the multiple standards necessary for economical industrial use. For example, algae grown as monocultures for biofuel production have not simultaneously and economically achieved high yields of the high-quality lipid-rich biomass desired for the industrial-scale production of bio-oil. Decades of study in the field of ecology have demonstrated that simultaneous increases in multiple functions, such as the quantity and quality of biomass, can occur in natural ecosystems by increasing biological diversity. Here, we show that species consortia of algae can improve the production of bio-oil, which benefits from both a high biomass yield and a high quality of biomass rich in fatty acids. We explain the underlying causes of increased quantity and quality of algal biomass among species consortia by showing that, relative to monocultures, species consortia can differentially regulate lipid metabolism genes while growing to higher levels of biomass, in part due to a greater utilization of nutrient resources. We identify multiple genes involved in lipid biosynthesis that are frequently upregulated in bicultures and further show that these elevated levels of gene expression are highly predictive of the elevated levels in biculture relative to that in monoculture of multiple quality metrics of algal biomass. These results show that interactions between species can alter the expression of lipid metabolism genes and further demonstrate that our understanding of diversity-function relationships from natural ecosystems can be harnessed to improve the production of bio-oil. IMPORTANCE Algal biofuels are one of the more promising forms of renewable energy. In our study, we investigate whether ecological interactions between species of microalgae regulate two important factors in cultivation-the biomass of the crop produced and the quality of the biomass that is produced. We found that species interactions often improved production yields, especially the fatty acid content of the algal biomass, and that differentially expressed genes involved in fatty acid metabolism are predictive of improved quality metrics of bio-oil. Other studies have found that diversity often improves productivity and stability in agricultural and natural ecosystems. Our results provide further evidence that growing multispecies crops of microalgae may improve the production of high-quality biomass for bio-oil.

This study determined the effect of a dried algae product containing beta-1,3-glucan on broiler performance and immunity during an Eimeria challenge. Heterotrophically grown Euglena gracilis, which contained ∼55% beta-1,3-glucan, was dried and milled for inclusion into a non-medicated starter diet. Two experiments were conducted to evaluate dietary treatments containing 0, 50, 100, 150, and 200 g/ton dried algae. In both experiments, male broilers were orally challenged on day 14 with a coccidial inoculum consisting of E. acervulina, E. maxima, and E. tenella. Fecal matter was collected 120-144 hours post-exposure to determine relative amounts of oocyte shedding and birds were sacrificed on day 20 for lesion scoring. Broiler performance was assessed on a weekly basis. In the first experiment, birds receiving dried algae at 50 and 200 g/ton showed a significant improvement in FCR compared to the infected control during the challenge period (days 14-20). In the second experiment, the dried algae treatment had no significant effect on FCR, but lesion scores were significantly reduced in the groups receiving 50, 150, and 200 g/ton dried algae relative to the infected control. In both experiments, the dried algae treatment did not significantly impact mortality or oocyte shedding. In the second experiment, staining of intestinal samples with fluorescently tagged antibodies demonstrated that dried algae at 100 g/ton increased the number of intestinal macrophages compared to the infected control. A significant and dose-dependent increase in intestinal MHC-II+ expression was also observed for birds fed dried algae, with an 8-fold increase observed in the 200 g/ton group relative to the infected control. Similarly, increased total immune cell density (measured by the mean fluorescence intensity of CD45+ cells) was also observed at 150 and 200 g/ton. Overall, these data suggest that dried algae rich in beta-1,3-glucan can help improve gut immunity and host protection, thereby reducing morbidity associated with coccidiosis.

This 42-day study evaluated the effects of dietary supplementation with β-1,3-glucan (Aleta™) on the vaccination response to Newcastle disease virus (NDV), avian infectious bronchitis virus (IBV), and infectious bursal disease (IBD) in a non-challenged environment. This trial included 600 chicks (all vaccinated with IBD at the hatchery) which were assigned to 1 of 3 treatments: vaccination (NDV, IBV), no vaccination, or vaccination combined with feed supplemented with Aleta (100 g/MT of feed). The vaccination with Aleta treatment group showed a trend for improved FCR that was not statistically significant. Control birds that were not vaccinated for IBV had significantly lower IBV titers on day 21 compared to birds that were vaccinated (both with and without Aleta). Surprisingly, there was significant separation among treatment groups for NDV titer levels, especially on day 21, where birds vaccinated and supplemented with Aleta had significantly higher titer levels compared to vaccination alone or no vaccination at all. Critically, only 14% of the birds receiving the vaccine plus Aleta had titer levels below the critical titer threshold for immunity compared to 28% of the birds receiving the vaccine alone and 40% of the unvaccinated birds. This suggests that Aleta supplementation may help to improve the vaccination response by birds, especially for NDV.

Euglena gracilis is a microalga capable of synthesizing various nutrients of interest in human and animal nutrition. When cultivated aerobically in the dark, Euglena synthesize paramylon, a storage polysaccharide comprised of high molecular weight beta-1,3-D-glucose polymers organized in cytoplasmic granules. Beta-glucans have been shown to have immune modulation effects, including anti-microbial, anti-tumor, and anti-oxidant properties, and metabolic effects, such as regulation of cholesterol and blood sugar levels. Preparations of E. gracilis and paramylon may therefore have potential utility as functional food ingredients for human and animal nutrition. A battery of toxicological studies was conducted on a dried preparation of E. gracilis and paramylon to support their safe food use. The dried alga was not genotoxic in a bacterial reverse mutation test and mammalian micronucleus test. In the subchronic toxicity study, rats were provided E. gracilis in the diet at levels of 0, 12,500, 25,000 or 50,000 ppm. Paramylon was provided at a concentration of 50,000 ppm. No effects that could be attributable to treatment were observed in clinical observations, body weight, food consumption, ophthalmology, hematology and clinical chemistry, urinalysis, and macroscopic and microscopic findings. A NOAEL of 50,000 ppm in the diet was determined for both ingredients.

Growing heterotrophic algae in reactors with sugar as the energy and carbon source rather than sunlight and carbon dioxide is an approach being commercialized today. However, the full environmental impacts of this fuel pathway have not been explored. The objective of this analysis was to compare the life cycle impacts of algal biodiesel produced heterotrophically to a phototrophic pathway featuring algae grown in ponds. A third, hybrid approach utilizing algae capable of both phototrophy and heterotrophy was also explored. Sugar beet and sugarcane were examined as feedstocks for the heterotrophic process. The results indicate that a reduction in the global warming potential (GWP) and an improvement in the net energy ratio (NER) for algal biodiesel could be possible for the heterotrophic and hybrid pathways relative to the phototroplaic, but only if reactor cultivation can be performed efficiently and with sugarcane as the feedstock For example, the NER varies from 0.6 to 1.6 for the heterotrophic pathway, depending on reactor performance, compared to 1.3 for the phototrophic pathway. Sugar crops used as feedstocks for heterotrophic cultivation present concerns about land constraints that are less of an issue for the phototrophic pathway. No pathway presented a clear advantage for the water stress impact metric.

We used hydrothermal carbonization (HTC) of wet algal bionias to facilitate.extraction of algal lipids that have value for both liquid biofuels and dietary supplements (i.e., nutraceuticals). HTC of the wet algal slurry produced a solid hydro char that retained more than 85% of the fatty acids in the original biomass and more than 75% of the valuable rimega-3 fatty acid eicosapentaerioic acid (EPA). The mass yield of hydro char was a linear function of the logarithm of the reaction ordinate, which is a measure of the severity of the carbonization conditions. The retained fatty acids could be readily extracted using ethanol, a renewable and food-grade solvent. A total fatty acid recovety of 74% was achieved through this combination of HTC and ethanol extraction. Thus, we herein, demonstrate a process for removing fatty-acid-containing lipids from Wet algae Using only water and a non-petroleum-derived solvent. The recovered lipids include both those titable for making biodiesel or green diesel and those suitable for the human diet.

Previous studies have demonstrated the feasibility of producing fatty-acid-derived hydrocarbons in Escherichia coli. However, product titers and yields remain low. In this work, we demonstrate new methods for improving fatty acid production by modifying central carbon metabolism and storing fatty acids in triacylglycerol. Based on suggestions from a computational model, we deleted seven genes involved in aerobic respiration, mixed-acid fermentation, and glyoxylate bypass (in the order of cyoA, nuoA, ndh, adhE, dld, pta, and iclR) to modify the central carbon metabolic/regulatory networks. These gene deletions led to increased total fatty acids, which were the highest in the mutants containing five or six gene knockouts. Additionally, when two key enzymes in the fatty acid biosynthesis pathway were over-expressed, we observed further increase in strain △cyoA△adhE△nuoA△ndh△pta△dld, leading to 202 mg/g dry cell weight of total fatty acids, ~250% of that in the wild-type strain. Meanwhile, we successfully introduced a triacylglycerol biosynthesis pathway into E. coli through heterologous expression of wax ester synthase/acyl-coenzyme:diacylglycerol acyltransferase (WS/DGAT) enzymes. The added pathway improved both the amount and fuel quality of the fatty acids. These new metabolic engineering strategies are providing promising directions for future investigation.

The high fertilizer demand for biodiesel production from microalgae is a significant challenge facing the commercialization of this promising technology. We investigated a processing strategy called hydrothermal carbonization (HTC) to convert wet algal biomass into a lipid‐rich hydrochar and aqueous phase (AP) co‐product. By reacting biomass at 200°C for 15 min, about 50% of the algae biomass became a solid hydrochar and roughly 40–70% of the C, N, and P in the reactant material dissolved into the AP. For the first time, an AP co‐product of this nature was analyzed by HPLC, GC‐MS and FT‐ICR‐MS to identify and characterize the dissolved organic matter. Using a unique marine bi‐culture suspected to contain a green algae ( Nannochloris ) and a cyanobacteria ( Synechocystis ), we demonstrated that this AP co‐product can support biomass growth better than a medium containing only inorganic nutrients. To manage unwanted contamination and optimize AP utilization, we employed a two‐stage growth process and fed‐batch additions of the AP co‐product. The effect of media recycling and nutrient supplementation, as well as a production model for a large‐scale facility, are discussed. Our work suggests that HTC can play a critical role in making algal biorefineries more sustainable by obviating biomass drying for fuel processing and recycling nutrients. © 2013 American Institute of Chemical Engineers Environ Prog, 32: 962–975, 2013

This work focuses on the production of biodiesel from wet, lipid-rich algal biomass using a two-step process involving hydrothermal carbonization (HTC) and supercritical in situ transesterification (SC-IST). Algal hydrochars produced by HTC were reacted in supercritical ethanol to determine the effects of reaction temperature, time, ethanol loading, water content, and pressure on the yield of fatty acid ethyl esters (FAEE). Reaction temperatures above 275 °C resulted in substantial thermal decomposition of unsaturated FAEE, thereby reducing yields. At 275 °C, time and ethanol loading had a positive impact on FAEE yield while increasing reaction water content and pressure reduced yields. FAEE yields as high as 79% with a 5:1 ethanol:fatty acid (EtOH:FA) molar ratio (150 min) and 89% with a 20:1 EtOH:FA molar ratio (180 min) were achieved. This work demonstrates that nearly all lipids within algal hydrochars can be converted into biodiesel through SC-IST with only a small excess of alcohol.

Microalgae have been investigated as a promising biodiesel feedstock; however, large-scale production is not currently cost-competitive with petroleum diesel, and its environmental impacts have received little attention. Using wastewater to supply nutrients for algal growth obviates synthetic fertilizer use, provides on-site nutrient removal, and reduces greenhouse gas emissions. In this work, anaerobically digested dairy manure was used to grow the oleaginous green alga Neochloris oleoabundans. In batch culture experiments with both synthetic media and anaerobic digester effluent, N. oleoabundans assimilated 90–95% of the initial nitrate and ammonium after 6 d and yielded 10–30% fatty acid methyl esters on a dry weight basis. Cellular lipid content and the N concentration in the growth media were inversely correlated. In addition, the proportion of polyunsaturated fatty acids (i.e. C16:3, C18:2, and C18:3) decreased with N concentration over time while the proportion of C18:1 fatty acid increased. Although N deficiency is likely the primary driver behind lipid accumulation, the influence of culture pH confounded results and requires further study. Other living microorganisms in the digester effluent were not observed to affect algal growth and lipid productivity, though the breakdown of organic nitrogen may have hindered lipid accumulation traditionally achieved through the manipulation of synthetic media. This work highlights the potential for waste-grown mono-algal cultures to produce high quality biodiesel while accomplishing simultaneous wastewater treatment.