Use of the ptxD gene as a portable selectable marker for chloroplast transformation in Chlamydomonas reinhardtii.

Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.

Biomass and lipid production from Chlorella vulgaris UTEX 26 cultivated in 2 m3 raceway ponds under semicontinuous mode during the spring season.

A Chlorella vulgaris UTEX 26 semicontinuous culture was implemented in 2000 L raceways with M medium during spring season at greenhouse conditions. Areal biomass productivities between 20 and 26 g m-2 d-1 were reached on the third day. The maximal areal lipid productivity obtained was 6.1 g m-2 d-1 and an increment in the saturated fatty acids (SFA) proportion (C14-C18) was favored in comparison with the fatty acids obtained with M medium in photobioreactors of 1 L and photoperiod light:darkness 12:12 h. After the eighth day of the culture or biomass concentrations above 0.25 g L-1, the microalgal cultures were prone to contamination by ciliates and amoebae, due to the sugars excreted by C. vulgaris UTEX 26. The periodical addition of NH4HCO3 to the microalgal culture maintained the ammonium concentration between 25 and 50 mg L-1, which contributed to diminish the contamination risks by protozoa.

Assessment of Chlorella vulgaris and indigenous microalgae biomass with treated wastewater as growth culture medium.

The aim of the present work was to evaluate the feasibility of microalgae cultivation using secondary treated domestic wastewater. Two Chlorella vulgaris strains (CICESE and UTEX) and an indigenous consortium, were cultivated on treated wastewater enriched with and without the fertilizer Bayfolan®. Biomass production for C. vulgaris UTEX, CICESE and the indigenous consortium grown in treated wastewater was 1.167±0.057, 1.575±0.434 and 1.125±0.250g/L, with a total lipid content of 25.70±1.24, 23.35±3.01and 20.54±1.23% dw, respectively. The fatty acids profiles were mainly composed of C16 and C18. Regardless of the media used, in all three strains unsaturated fatty acids were the main FAME (fatty acids methyl esters) accumulated in a range of 45-62%. An enrichment of treated wastewater with Bayfolan® significantly increased the production of biomass along with an increase in pigments and proteins of ten and threefold, respectively.

Biomass and lipid production from Nannochloropsis oculata growth in raceway ponds operated in sequential batch mode under greenhouse conditions.

The effect of sequential batch cultures of the marine microalgae Nannochloropsis oculata on lipid and biomass production was studied in 200-L raceway ponds for 167 days (nine harvesting cycles) during winter and spring seasons under greenhouse conditions. The highest biomass concentration and productivity were 1.2 g/L and 49.8 mg/L/day on days 73 (5th cycle) and 167 (9th cycle), respectively. The overall interval of lipid production was between 131 and 530 mg/L. Despite the daily and seasonal variations of light irradiance (0-1099 µmol photon/m2 s), greenhouse temperature (2.1-50.7 °C), and culture temperature (12.5-31.4 °C), ANOVA analysis showed no statistical difference (p value > 0.01) on the fatty acid methyl ester (FAMES) composition over the nine harvesting cycles evaluated. The most abundant FAMES were palmitic (C16:0), stearic (C18:0) and palmitoleic (C16:1∆9) acids with 37.1, 28.6, and 8.4 %, respectively. The sequential batch cultures of N. oculata in raceway ponds showed an increasing biomass production in each new cycle while keeping the quality of the fatty acid mixture under daily and seasonal variations of light irradiance and temperature.

A novel culture medium designed for the simultaneous enhancement of biomass and lipid production by Chlorella vulgaris UTEX 26.

A novel culture medium to enhance the biomass and lipid production simultaneously by Chlorella vulgaris UTEX 26 was designed in three stages of optimization. Initially, a culture medium was inferred applying the response surface method to adjust six factors [NaNO3, NH4HCO3, MgSO4·7H2O, KH2PO4, K2HPO4 and (NH4)2HPO4], which were selected on the basement of BBM (Bold's Basal Medium) and HAMGM (Highly Assimilable Minimal Growth Medium) culture media. Afterwards, the nitrogen source compound was optimized to reduce both, ammonium and nitrate concentrations. As result of the optimization process, the proposed culture medium improved 40% the biomass (0.73gL(-1)) compared with the BBM medium and 85% the lipid concentration (281mgL(-1)), with respect to HAMGM medium. Some culture media components concentrations were reduced up to 50%. Gas chromatography analysis revealed that C16:0, C18:0, C18:1, C18:2 and C18:3 were the major fatty acids produced by C. vulgaris UTEX 26.

Petroleum-influenced beach sediments of the Campeche Bank, Mexico: diversity and bacterial community structure assessment.

The bacterial diversity and community structure were surveyed in intertidal petroleum-influenced sediments of ≈ 100 km of a beach, in the southern Gulf of Mexico. The beach was divided in twenty sampling sites according to high, moderate and low petroleum influence. Densities of cultured heterotrophic (HAB) and hydrocarbon degrading bacteria (HDB) were highly variable in sediments, with little morphological assortment in colonies. PCR-RISA banding patterns differentiated distinct communities along the beach, and the bacterial diversity changed inversely to the degree of petroleum hydrocarbon influence: the higher TPH concentration, the lower genotype diversity. Seven DNA sequences (Genbank EF191394 -EF191396 and EF191398 -EF191401) were affiliated to uncultured members of Gemmatimonas, Acidobacterium, Desulfobacteraceae, Rubrobacterales, Actinobacterium and the Fibrobacteres/Acidobacteria group; all the above taxa are known for having members with active roles in biogeochemical transformations. The remaining sequences (EF191388 - EF191393 and EF191397) affiliated to Pseudoalteromonas, and to oil-degrading genera such as Pseudomonas, Vibrio and Marinobacter, being the last one an obligate oil-degrading bacterium. An exchange of bacteria between the beach and the oil seep environment, and the potential cleaning-up role of bacteria at the southern Gulf of Mexico are discussed.

Bacterial diversity of marine seeps in the southeastern Gulf of Mexico.

The diversity of bacterial communities of shallow (< or = 100 m depth) oil seep marine sediments from the southern Gulf of Mexico was evaluated. The geochemical properties of seep sediments were characterized as well as their microbial diversity in oil seep and control sediments. Bacteria were identified through molecular tools as belonging to the genera Marinobacter, Idiomarina, Marinobacterium, Frauteria and an unknown bacterium. Bacteria might be important components of microbial communities in Total Petroleum Hydrocarbon (TPH)-containing environments, displaying either facultative metabolism or able to grow only in petroleum-containing media. The identification of bacteria in shallow oil seep sediments could be used as indicators of marine hydrocarbons in southeastern Gulf of Mexico.

Encystment of Azotobacter nigricans grown diazotrophically on kerosene as sole carbon source.

Encystment of Azotobacter nigricans was induced by its diazotrophic cultivation on kerosene. Its growth and nitrogenase activity were affected by kerosene in comparison to cultures grown on sucrose. Electron microscopy of vegetative cells showed that when nitrogenase activity was higher and the poly-beta-hydroxybutyrate granules were not present to a significant extent, peripheral bodies were abundant. After 8 days of culture on kerosene, the presence of cysts with intracellular bunches of poly-beta-hydroxybutyrate granules was observed. Germination of cysts bears germinating multicelled yet unbroken capsule cysts with up to three cells inside. This is the first report of encystment induction of Azotobacter species grown on kerosene.

Feasibility study of bioremediation of a drilling-waste-polluted soil: stimulation of microbial activities and hydrocarbon removal.

The objective of this study was to determine the feasibility of bioremediation as a treatment option for an aged and chronically polluted drilling waste soil located at the Southeast of Mexico. The polluted drilling-waste site with a mean total petroleum hydrocarbon concentration (TPHs) of 39,397 +/- 858 mg/kg was treated with one dose of a nutrient-surfactant commercial product at 40 mg/kg soil and two doses of H2O2 (50 and 100 mg H2O2/kg soil). In this study, the parameters that were monitored include soil respiration, heterotrophic and hydrocarbon-degrading bacteria as biological indicators, catalase and dehydrogenase activities, and TPHs degradation as decontamination parameters. The results demonstrated that the microbial activities can be stimulated in a polluted drilling-waste site by the addition of H2O2 and commercial product, thereby resulting in increasing TPHs degradation. These aspects must be taken into account when biodegradation studies involve the application of a commercial product.