Morphological characterization, growth appraisal, and probing biofuels potential of newly isolated Scenedesmus sp. from desert Cholistan.

Microalgae have an excellent potential for producing valuable natural products, including biofuels. Therefore, it is imperative to explore and document the existing microalgal flora and utilize their potentials to cope the increasing human needs. The present work aims at exploring and characterizing newly isolated microalgae from desert Cholistan, a habitat with myriad algal diversity. Light microscopy, scanning electron microscopy, and molecular phylogenetic approaches were used for species-level identification. Characterization and growth optimization of Scendesmus sp. were analyzed under three different growth modes to determine the most favorable conditions for increasing biomass, growth rate, and lipid content. The results revealed that mixotrophic (MT) mode significantly increases photosynthetic activity, growth rate, and lipid content with glycerol as supplement carbon source. The investigated Scenedesmus dimorphous produced a maximum dry weight of 1.73 g L-1 , improved fatty acid methyl esters profile and yield lipid up to 40% of DCW (68 g L-1 ) under MT mode, which is almost double to that of photoautotrophic cultivation. The glycerol availability in medium has been identified as the critical element for boosting growth and lipid content. Thus, it can reduce the cost of biofuel production.

Demonstration and optimization of sequential microaerobic dark- and photo-fermentation biohydrogen production by immobilized Rhodobacter capsulatus JP91.

Hydrogen generation from complex substrates composed of simple sugars has the potential to mitigate future worldwide energy demand. The biohydrogen potential of a sequential microaerobic dark- and photo-fermentative system was investigated using immobilized Rhodobacter capsulatus JP91. Biological hydrogen production from glucose was carried out using a batch process and a bench-scale bioreactor. Response surface methodology with a Box-Behnken design was employed to optimize key parameters such as inoculum concentration, oxygen concentration, and glucose concentration. The maximum hydrogen production (21 ±â€¯0.25 mmol H2/L) and yield (7.8 ±â€¯0.1 mol H2/mol glucose) were obtained at 6 mM glucose, 4.5% oxygen and 62.5 v/v% inoculum concentration, demonstrating the feasibility of enhanced hydrogen production by immobilized R. capsulatus JP91 in a sequential system. This is the first time that a sequential process using an immobilized system has been described. This system also achieved the highest hydrogen yield obtained by an immobilized system so far.

Optimization of the yield of dark microaerobic production of hydrogen from lactate by Rhodopseudomonas palustris.

Hydrogen yields of dark fermentation are limited due to the need to also produce reduced side products, and photofermentation, an alternative, is limited by the need for light. A relatively new strategy, dark microaerobic fermentation, could potentially overcome both these constraints. Here, application of this strategy demonstrated for the first time significant hydrogen production from lactate by a single organism in the dark. Response surface methodology (RSM) was used to optimize substrate and oxygen concentration as well as inoculum using both (1) regular batch and (2) O2 fed batch cultures. The highest hydrogen yield (HY) was observed under regular batch (1.4±0.1molH2/mollactate) and the highest hydrogen production (HP) (173.5µmolH2) was achieved using O2 fed batch. This study has provided proof of principal for the ability of microaerobic fermentation to drive thermodynamically difficult reactions, such as the conversion of lactate to hydrogen.

Hydrogen Production by a Chlamydomonas reinhardtii Strain with Inducible Expression of Photosystem II.

Chlamydomonas reinhardtii cy6Nac2.49 is a genetically modified algal strain that activates photosynthesis in a cyclical manner, so that photosynthesis is not active constitutively in the presence of oxygen, but is turned on only in response to a metabolic trigger (anaerobiosis). Here, we further investigated hydrogen production by this strain comparing it with the parental wild-type strain under photoheterotrophic conditions in regular tris-acetate-phosphate (TAP) medium with a 10-h:14-h light/dark regime. Unlike the wild-type, whose level of H2 production remained low during illumination, H2 production in the mutant strain increased gradually with each subsequent light period, and by the final light period was significantly higher than the wild-type. The relatively low Photosystem II (PSII) activity of the mutant culture was shown by low fluorescence yield both in the dark (Fv/Fm) and in the light (δF/Fm') periods. Measurement of oxygen evolution confirmed the low photosynthetic activity of the mutant cells, which gradually accumulated O2 to a lesser extent than the wild-type, thus allowing the mutant strain to maintain hydrogenase activity over a longer time period and to gradually accumulate H2 during periods of illumination. Therefore, controllable expression of PSII can be used to increase hydrogen production under nutrient replete conditions, thus avoiding many of the limitations associated with nutrient deprivation approaches sometimes used to promote hydrogen production.

Effect of nitrogen regime on microalgal lipid production during mixotrophic growth with glycerol.

Mixotrophic growth of microalgae to boost lipid production is currently under active investigation. Such a process could be of practical importance if a cheap source of organic carbon, such as waste glycerol from biodiesel production, could be used. Several previous studies have already demonstrated that this carbon source can be used by different indigenous strains of microalgae. In this study it is shown that different nitrogen limitation strategies can be applied to further increase lipid production during growth with glycerol. In one strategy, cultures were grown in nitrogen replete medium and then resuspended in nitrogen free medium. In a second strategy, cultures were grown with different initial concentrations of nitrate. Lipid production by the two microalgal strains used, Chlorella sorokiniana (PCH02) and Chlorella vulgaris (PCH05), was shown to be boosted by strategies of nitrogen limitation, but they responded differently to how nitrogen limitation was imposed.

Draft Genome Sequence of the Photoheterotrophic Chloracidobacterium thermophilum Strain OC1 Found in a Mat at Ojo Caliente.

Metagenomics of an enrichment culture from a New Mexico hot spring allowed the description of a draft genome of a Chloracidobacterium thermophilum strain for the first time outside Yellowstone National Park with a surprisingly high degree of identity with the type strain.

Draft Genome Sequence of a Thermophilic Cyanobacterium from the Family Oscillatoriales (Strain MTP1) from the Chalk River, Colorado.

The draft genome (57.7% GC, 7,647,882 bp) of the novel thermophilic cyanobacterium MTP1 was determined by metagenomics of an enrichment culture. The genome shows that it is in the family Oscillatoriales and encodes multiple heavy metal resistances as well as the capacity to make exopolysaccharides.

Strain variation in microalgal lipid production during mixotrophic growth with glycerol.

Algal cultivation at high latitudes is challenged by the relatively low annual solar flux. One possible scenario to overcome this limitation is the use of mixotrophic growth to potentially boost biomass and lipid production. Here the effect of glycerol addition on the growth and lipid production by twelve indigenous microalgae was examined. The results show that there is considerable strain dependent variation in the maximum growth rate under mixotrophic conditions with the addition of glycerol causing in some cases up to a 2.4-fold increase in growth rate and a up to a 1.9-fold increase in biomass. In addition, glycerol increased total lipid production 40-60% in some strains. These results also show the value in screening culture collections for desired traits independent of strain identification since here one (PCH02) of the five Chlorella strains showed a large increase in lipid with glycerol.

Heterologous expression of proteorhodopsin enhances H2 production in Escherichia coli when endogenous Hyd-4 is overexpressed.

Proteorhodopsin (PR) is a light harvesting protein widely distributed among bacterioplankton that plays an integral energetic role in a new pathway of marine light capture. The conversion of light into chemical energy in non-chlorophyll-based bacterial systems could contribute to overcoming thermodynamic and metabolic constraints in biofuels production. In an attempt to improve biohydrogen production yields, H2 evolution catalyzed by endogenous hydrogenases, Hyd-3 and/or Hyd-4, was measured when recombinant proteorhodopsin (PR) was concomitantly expressed in Escherichia coli cells. Higher amounts of H2 were obtained with recombinant cells in a light and chromophore dependent manner. This effect was only observed when HyfR, the specific transcriptional activator of the hyf operon encoding Hyd-4 was overexpressed in E. coli, suggesting that an excess of protons generated by PR activity could increase hydrogen production by Hyd-4 but not by Hyd-3. Although many of the subunits of Hyd-3 and Hyd-4 are very similar, Hyd-4 possesses three additional proton-translocating NADH-ubiquinone oxidoreductase subunits, suggesting that it is dependent upon ΔµH(+). Altogether, these results suggest that protons generated by proteorhodopsin in the periplasm can only enhance hydrogen production by hydrogenases with associated proton translocating subunits.

Utilization of biodiesel-derived glycerol or xylose for increased growth and lipid production by indigenous microalgae.

Microalgae are a promising alternative for sustainable biofuel production, but production yields and costs present a significant bottleneck. Here, the use of glycerol and xylose to boost the lipid yield was evaluated using ten strains from the Université de Montréal collection of microalgae. This report shows that some microalgal strains are capable of mixotrophic and heterotrophic growth on xylose, the major carbon source found in wastewater streams from pulp and paper industries, with an increase in growth rate of 2.8-fold in comparison to photoautotrophic growth, reaching up to µ=1.1/d. On glycerol, growth rates reached as high as µ=1.52/d. Lipid productivity increased up to 370% on glycerol and 180% on xylose for the strain LB1H10, showing the suitability of this strain for further development of biofuels production through mixotrophic cultivation.

Biological reformation of ethanol to hydrogen by Rhodopseudomonas palustris CGA009.

A future hydrogen economy requires the sustainable production of renewable hydrogen. One method to produce hydrogen from stored renewable energy could be through reformation of bioethanol. However, chemically catalyzed reformation processes, although well studied, still present a number of significant technical challenges. Here, bioreformation of ethanol to hydrogen by photofermentation with the photosynthetic bacterium Rhodopseudomonas palustris is described. Cultures were shown to tolerate up to 2% ethanol. An RSM (response surface methodology) was carried out in which three key factors, light intensity, and ethanol and glutamate concentrations were varied. The results showed that nearly 2mol of H2 could be obtained from one mole of ethanol, 33% of that theoretically possible.

The use of Design of Experiments and Response Surface Methodology to optimize biomass and lipid production by the oleaginous marine green alga, Nannochloropsis gaditana in response to light intensity, inoculum size and CO2.

Biodiesel produced from microalgal lipids is being considered as a potential source of renewable energy. However, a number of hurdles will have to be overcome if such a process is to become practical. One important factor is the volumetric production of biomass and lipid that can be achieved. The marine alga Nannochloropsis gaditana is under study since it is known to be highly oleaginous and has a number of other attractive properties. Factors that might be important in biomass and lipid production by this alga are light intensity, inoculum size and CO2. Here we have carried out for the first time a RSM-DOE study of the influence of these important culture variables and define conditions that maximize biomass production, lipid content (BODIPY® fluorescence) and total lipid production. Moreover, flow cytometry allowed the examination on a cellular level of changes that occur in cellular populations as they age and accumulate lipids.

Building a better mousetrap I: using Design of Experiments with unconfounded ions to discover superior media for growth and lipid production by Chlorella sp. EN1234.

An unconfounded Scheffe Mix approach was used to probe important ions and their interactions in supporting biomass and lipid production by Chlorella sp. EN1234. Six major cations and anions; NH4(+), NO3(-), Na(+), K(+) PO4(-) and Cl(-) were examined. Piepel plots and RSM analysis showed that in a number of cases, the major media anions PO4(-) and Cl(-) negatively influence final cell densities, and that maximal cell density is obtained with nitrate over ammonium, with an optimal effect when mixed with equal molar potassium. As well, although it is commonly assumed that lipid content increases in nitrogen deficient media, here little correlation between nitrogen content and total lipid content was found with mixtures that supported high lipid productivity. Thus these mixtures define the composition space within which further R&D might produce the best trade-off between total biomass production and high cellular lipid content.

Building a better mousetrap II: using Design of Experiments with unconfounded ions to compare the growth of different microalgae.

A large number of unconfounded media variations were used with a Scheffe Mix Model to examine in an unambiguous fashion the effects of variations in six important ions; NH4(+), NO3(-), Na(+), K(+), PO4(-), and Cl(-), on the growth of Chlorella vulgaris. This allows several novel observations on media components, for example, the inhibitory effects of chloride, to be made. Using a side by side comparison, it is shown that two strains of Chlorella show significant physiological and functional differences brought out by this approach. Testing selected formulations with a diverse set of algae demonstrated different effects on both growth and cellular lipid content, in some cases driving significant lipid production. This suggests that future work using a larger portion of media composition space could lead to the development of novel media supporting maximal biomass production and lipid production.

Enhanced photo-fermentative H2 production using Rhodobacter sphaeroides by ethanol addition and analysis of soluble microbial products.

BACKGROUND: Biological fermentation routes can provide an environmentally friendly way of producing H2 since they use renewable biomass as feedstock and proceed under ambient temperature and pressure. In particular, photo-fermentation has superior properties in terms of achieving high H2 yield through complete degradation of substrates. However, long-term H2 production data with stable performance is limited, and this data is essential for practical applications. In the present work, continuous photo-fermentative H2 production from lactate was attempted using the purple non-sulfur bacterium, Rhodobacter sphaeroides KD131. As a gradual drop in H2 production was observed, we attempted to add ethanol (0.2% v/v) to the medium. RESULTS: As continuous operation went on, H2 production was not sustained and showed a negligible H2 yield (< 0.5 mol H2/mol lactateadded) within two weeks. Electron balance analysis showed that the reason for the gradual drop in H2 production was ascribed to the increase in production of soluble microbial products (SMPs). To see the possible effect of ethanol addition, a batch test was first conducted. The presence of ethanol significantly increased the H2 yield from 1.15 to 2.20 mol H2/mol lactateadded, by suppressing the production of SMPs. The analysis of SMPs by size exclusion chromatography showed that, in the later period of fermentation, more than half of the low molecular weight SMPs (< 1 kDa) were consumed and used for H2 production when ethanol had been added, while the concentration of SMPs continuously increased in the absence of ethanol. It was found that the addition of ethanol facilitated the utilization of reducing power, resulting in an increase in the cellular levels of NAD(+) and NADP(+). In continuous operation, ethanol addition was effective, such that stable H2 production was attained with an H2 yield of 2.5 mol H2/mol lactateadded. Less than 15% of substrate electrons were used for SMP production, whereas 35% were used in the control. CONCLUSIONS: We have found that SMPs are the key factor in photo-fermentative H2 production, and their production can be suppressed by ethanol addition. However, since external addition of ethanol to the medium represents an extra economic burden, ethanol should be prepared in a cost-effective way.

Screening microalgae native to Quebec for wastewater treatment and biodiesel production.

Biodiesel production from microalgae lipids is being considered as a potential source of renewable energy. However, practical production processes will probably require the use of local strains adapted to prevailing climatic conditions. This report describes the isolation of 100 microalgal strains from freshwater lakes and rivers located in the vicinity of Montreal, Quebec, Canada. Strains were identified and surveyed for their growth on secondary effluent from a municipal wastewater treatment plant (La Prairie, QC, Canada) using a simple and high throughput microalgal screening method employing 12 well plates. The biomass and lipid productivity of these strains on wastewater were compared to a synthetic medium under different temperatures (10±2°C and 22±2°C) and a number identified that showed good growth at 10°C, gave a high lipid content (ranging from 20% to 45% of dry weight) or a high capacity for nutrient removal.

Characterization of growth and lipid production by Chlorella sp. PCH90, a microalga native to Quebec.

Microalgae are being investigated as potential candidates for biodiesel production since they can be grown without competition with food production, have an inherently fast growth rate, and can have a high lipid content under different nutrient limiting conditions. However, large scale production will best be carried out with indigenous strains, well adapted to local conditions. This study reports on the characterization of the novel microalga Chlorella sp. PCH90, isolated in Quebec. Its molecular phylogeny was established and lipid production studies as a function of the initial concentrations of nitrate, phosphate, and sodium chloride were carried out using response surface methodology. Under the appropriate conditions this microalga could produce up to 36% lipid and grew well in both synthetic medium and secondary effluent from a wastewater treatment plant at both 22 and 10°C. Thus, this strain is promising for further development as a potential biofuels producer under local climatic conditions.

Addressing the challenges for sustainable production of algal biofuels: I. Algal strains and nutrient supply.

Microalgae hold promise for the production of sustainable replacement of fossil fuels due to their high growth rates, ability to grow on non-arable land and their high content, under the proper conditions, of high energy compounds that can be relatively easily chemically converted to fuels using existing technology. However, projected large-scale algal production raises a number of sustainability concerns concerning land use, net energy return, water use and nutrient supply. The state-of-the-art of algal production of biofuels is presented with emphasis on some possible avenues to provide answers to the sustainability questions that have been raised. Here, issues concerning algal strains and supply of nutrients for large-scale production are discussed. Since sustainability concerns necessitate the use of wastewaters for supply of bulk nutrients, emphasis is placed on the composition and suitability of different wastewater streams. At the same time, algal cultivation has proven useful in waste treatment processes, and thus this aspect is also treated in some detail.

Addressing the challenges for sustainable production of algal biofuels: II. Harvesting and conversion to biofuels.

In order to ensure the sustainability of algal biofuel production, a number of issues need to be addressed. Previously, we reviewed some of the questions in this area involving algal species and the important challenges of nutrient supply and how these might be met. Here, we take up issues involving harvesting and the conversion ofbiomass to biofuels. Advances in both these areas are required if these third-generation fuels are to have a sufficiently high net energy ratio and a sustainable footprint. A variety of harvesting technologies are under investigation and recent studies in this area are presented and discussed. A number of different energy uses are available for algal biomass, each with their own advantages as well as challenges in terms of efficiencies and yields. Recent advances in these areas are presented and some of the especially promising conversion processes are highlighted.