TOR kinase activity in Chlamydomonas reinhardtii is modulated by cellular metabolic states.

Target of rapamycin (TOR) kinase is a sensor and a central integrator of internal and external metabolic cues. However, in algae and in higher plants, the components of TOR kinase signaling are yet to be characterized. Here, we establish an assay system to study TOR kinase activity in Chlamydomonas reinhardtii using the phosphorylation status of its putative downstream target, CrS6K. Using this assay, we probe the modulation of cellular TOR kinase activity under various physiological states such as photoautotrophy, heterotrophy, mixotrophy, and nitrogen (N) starvation. Importantly, we uncover that excess acetate in the medium leads to high cellular reactive oxygen species levels, triggering autophagy and a concomitant drop in TOR kinase activity in a dose-dependent manner, thus leading to a N-starvation-like cellular phenotype, even when nitrogen is present.

New findings on the effect of glyphosate on autotrophic and heterotrophic picoplankton structure: A microcosm approach.

Massive use of glyphosate-based herbicides in agricultural activities has led to the appearance of this herbicide in freshwater systems, which represents a potential threat to these systems and their communities. These herbicides can affect autotrophic and heterotrophic picoplankton abundance. However, little is known about glyphosate impact on the whole structure of these assemblages. Herein, we used an 8-day long microcosm approach under indoor controlled conditions to analyze changes in the structure of picoplankton exposed to a single pulse of glyphosate. The analyzed picoplankton correspond to two outdoor ponds with contrasting states: "clear" (chlorophyll-a = 3.48 µg L-1± 1.15; nephelometric turbidity, NTU = 1) and "turbid" (chlorophyll-a = 105.96 µg L-1 ± 15.3; NTU = 48). We evaluated herbicide impact on different picoplankton cytometric populations and further explored changes in bacterial dominant operational taxonomic units (OTUs) fingerprinting. We observed that glyphosate induced a drastic decrease in the abundance of phycocyanin-rich picocyanobacteria. Particularly, in the turbid system this effect resulted in an 85 % decrease in the abundance of the whole autotrophic picoplankton. Glyphosate also changed the structure of the heterotrophic fraction by means of changing bacterial dominant OTUs fingerprinting patterns in both systems and by shifting the relative abundances of cytometric groups in the clear scenario. These results demonstrate that upon glyphosate exposure picoplanktonic fractions face not only the already reported changes in abundance, but also alterations in the composition of cytometric groups and of bacterial dominant operational taxonomic units. This research provides suitable and still little explored tools to analyze agrochemical effects on picoplanktonic communities.

The industrial yeast Pichia pastoris is converted from a heterotroph into an autotroph capable of growth on CO2.

The methylotrophic yeast Pichia pastoris is widely used in the manufacture of industrial enzymes and pharmaceuticals. Like most biotechnological production hosts, P. pastoris is heterotrophic and grows on organic feedstocks that have competing uses in the production of food and animal feed. In a step toward more sustainable industrial processes, we describe the conversion of P. pastoris into an autotroph that grows on CO2. By addition of eight heterologous genes and deletion of three native genes, we engineer the peroxisomal methanol-assimilation pathway of P. pastoris into a CO2-fixation pathway resembling the Calvin-Benson-Bassham cycle, the predominant natural CO2-fixation pathway. The resulting strain can grow continuously with CO2 as a sole carbon source at a µmax of 0.008 h-1. The specific growth rate was further improved to 0.018 h-1 by adaptive laboratory evolution. This engineered P. pastoris strain may promote sustainability by sequestering the greenhouse gas CO2, and by avoiding consumption of an organic feedstock with alternative uses in food production.

Optimization of heterotrophic cultivation of Chlorella sp. HS2 using screening, statistical assessment, and validation.

The heterotrophic cultivation of microalgae has a number of notable advantages, which include allowing high culture density levels as well as enabling the production of biomass in consistent and predictable quantities. In this study, the full potential of Chlorella sp. HS2 is explored through optimization of the parameters for its heterotrophic cultivation. First, carbon and nitrogen sources were screened in PhotobioBox. Initial screening using the Plackett-Burman design (PBD) was then adopted and the concentrations of the major nutrients (glucose, sodium nitrate, and dipotassium phosphate) were optimized via response surface methodology (RSM) with a central composite design (CCD). Upon validation of the model via flask-scale cultivation, the optimized BG11 medium was found to result in a three-fold improvement in biomass amounts, from 5.85 to 18.13 g/L, in comparison to a non-optimized BG11 medium containing 72 g/L glucose. Scaling up the cultivation to a 5-L fermenter resulted in a greatly improved biomass concentration of 35.3 g/L owing to more efficient oxygenation of the culture. In addition, phosphorus feeding fermentation was employed in an effort to address early depletion of phosphate, and a maximum biomass concentration of 42.95 g/L was achieved, with biomass productivity of 5.37 g/L/D.

Bacterial community shifts evaluation in the sediments of Puyang River and its nitrogen removal capabilities exploration by resuscitation promoting factor.

Identifying indigenous bacterial community and exploring the potential of native microorganisms are crucial for in situ bioremediation of nitrogenous pollutants in water bodies. This study evaluated the bacterial communities of sediment samples from a nitrogen polluted river, and revealed the possible environmental factors shaping the bacterial populations. Importantly, viable but non-culturable bacteria which possessed nitrogen removal capabilities in indigenous population of the sediments were explored by resuscitation promoting factor (Rpf). It was found that the sediments from upstream (URS) and lower stream (LRS) of Puyang river showed both different pollutants levels and bacterial community. Nitrate nitrogen, organic carbon and ammonium nitrogen probably had a significant effect on bacterial compositions between URS and LRS. From URS and LRS, a total of thirteen strains with heterotrophic nitrification ability were resuscitated by Rpf addition, which belonged to genera Bacillus, Pseudomonas, Stenotrophomonas and Acinetobacter. Among them, the strain Pseudomonas sp. SSPR1 was found to display high removal capabilities of simultaneous nitrification and denitrification, and the average ammonium and nitrate removal rates were 2.23 and 0.86 mg/(L·h), respectively. These resuscitated strains could be considered to be used for biological nitrogen removal in rivers and their receiving water bodies.

Silver stress differentially affects growth of phototrophic and heterotrophic chrysomonad flagellate populations.

Silver ions are among the predominant anthropogenic introduced pollutants in aquatic systems. As silver has effects on species at all trophic levels the community composition in aquatic habitats can be changed as a result of silver stress. The response of planktonic protists to environmental stressors is particularly important as they act both as producers and consumers in complex planktonic communities. Chrysomonad flagellates are of major interest, since this group includes heterotrophic, mixotrophic and phototrophic taxa, and therefore allows analysis of silver stress in organisms with contrasting nutritional strategies independent of a potential taxonomic bias. In a series of lab experiments, we compared the response of different trophic chrysophyte strains to low (5 µg L-1), medium (10 µg L-1) and high (20 µg L-1) nominal Ag concentrations in combination with changes in temperature and light intensity (phototrophs), temperature and food concentration (heterotrophs), or a combination of the above settings (mixotrophs). All tested strains were negatively affected by silver in their growth rates. The phototrophic strains reacted strongly to silver stress, whereas light intensity and temperature had only minor effects on growth rates. For heterotrophic strains, high food concentration toned down the effect of silver, whereas temperatures outside the growth optimum had a combined stress effect. The mixotrophic strains reacted differently depending on whether their nutritional mode was dominated by heterotrophy or by phototrophy. The precise response pattern across all variables was uniquely different for every single species we tested. The present work contributes to a deeper understanding of the effects of environmental stressors on complex planktonic communities. It indicates that silver will negatively impact planktonic communities and may create shifts in their composition and functioning.

Enhancement of coral calcification via the interplay of nickel and urease.

Corals are the main reef builders through the formation of calcium carbonate skeletons. In recent decades, coral calcification has however been impacted by many global (climate change) and local stressors (such as destructive fishing practices and changes in water quality). In this particular context, it is crucial to identify and characterize the various factors that promote coral calcification. We thus performed the first investigation of the effect of nickel and urea enrichment on the calcification rates of three coral species. These two factors may indeed interact with calcification through the activity of urease, which catalyzes the hydrolysis of urea to produce inorganic carbon and ammonia that are involved in the calcification process. Experiments were performed with the asymbiotic coral Dendrophyllia arbuscula and, to further assess if urea and/or nickel has an indirect link with calcification through photosynthesis, results were compared with those obtained with two symbiotic corals, Acropora muricata and Pocillopora damicornis, for which we also measured photosynthetic rates. Ambient and enriched nickel (0.12 and 3.50 µg L-1) combined with ambient and enriched urea concentrations (0.26 and 5.52 µmol L-1) were tested during 4 weeks in aquaria. We demonstrate in the study that a nickel enrichment alone or combined with a urea enrichment strongly stimulated urea uptake rates of the three tested species. In addition, this enhancement of urea uptake and hydrolysis significantly increased the long-term calcification rates (i.e. growth) of the three coral species investigated, inducing a 1.49-fold to 1.64-fold increase, respectively for D. arbuscula and P. damicornis. Since calcification was greatly enhanced by nickel in the asymbiotic coral species - i.e. in absence of photosynthesis - we concluded that the effect of increased urease activity on calcification was mainly direct. According to our results, it can be assumed that corals in some fringing reefs, benefiting from seawater enriched in nickel may have advantages and might be able to use urea more effectively as a carbon and nitrogen source. It can also be suggested that urea, for which hotspots are regularly measured in reef waters may alleviate the negative consequences of thermal stress on corals.

Dissolved organic carbon leaching from plastics stimulates microbial activity in the ocean.

Approximately 5.25 trillion plastic pieces are floating at the sea surface. The impact of plastic pollution on the lowest trophic levels of the food web, however, remains unknown. Here we show that plastics release dissolved organic carbon (DOC) into the ambient seawater stimulating the activity of heterotrophic microbes. Our estimates indicate that globally up to 23,600 metric tons of DOC are leaching from marine plastics annually. About 60% of it is available to microbial utilization in less than 5 days. If exposed to solar radiation, however, this DOC becomes less labile. Thus, plastic pollution of marine surface waters likely alters the composition and activity of the base of the marine food webs. It is predicted that plastic waste entering the ocean will increase by a factor of ten within the next decade, resulting in an increase in plastic-derived DOC that might have unaccounted consequences for marine microbes and for the ocean system.

Aerobic-heterotrophic nitrogen removal through nitrate reduction and ammonium assimilation by marine bacterium Vibrio sp. Y1-5.

An aerobic marine bacterium Vibrio sp. Y1-5 was screened to achieve efficient nitrate and ammonium removal simultaneously and fix nitrogen in cells without N loss. Approximately 98.0% of nitrate (100mg/L) was removed in 48h through assimilatory nitrate reduction and nitrate reductase was detected in the cytoplasm. Instead of nitrification, the strain assimilated ammonium directly, and it could tolerate as high as 1600mg/L ammonium concentration while removing 844.6mg/L. In addition, ammonium assimilation occurred preferentially in the medium containing nitrate and ammonium with a total nitrogen (TN) removal efficiency of 80.4%. The results of nitrogen balance and Fourier infrared spectra illustrated that the removed nitrogen was all transformed to protein or stored as organic nitrogen substances in cells and no N was lost in the process. Toxicological studies with the brine shrimp species Artemia naupliia indicated that Vibrio sp. Y1-5 can be applied in aquatic ecosystems safely.

Multi metal assessment on biofilm formation in offshore environment.

Eight metal and non-metal coupons were exposed to seawater in offshore platform laboratory on biofilm formation in seven days of every month interval time for one year period. Among metal coupons, the highest mean heterotrophic biofilm bacteria population (HBBP) ranges were observed in mild steel (MS) coupon (33.03 to 40.00 [×104] CFUscm-2) and lowest in copper (Cu) coupon (3.0 to 5.1 [×104] CFUscm-2). Non-metal coupons show, highest mean HBBP ranges in glass (GL) coupon (29.6 to 66.0 [×104] CFUscm-2) and lowest in polyvinyl chloride (PVC) coupon (34.4 to 52.5 [×104] CFUscm-2). Within the studied coupons, high HBBP level was witnessed in GL and simultaneously low in Cu due to its toxic leaching product of Cu2O. Gram-positive bacterial deposit show dominated trend in all coupons with increasing order of Cu>SS>MS>PVC>GL>Cu-Ni 70/30>Cu-Ni 90/10>Br and Gram-negative show different in the order of Br>Cu-Ni 90/10>Cu-Ni 70/30>GL>PVC>MS>SS>Cu, respectively. The 16S rDNA sequence confirmed, heterotrophic biofilm Bacillus sp. (Gram-positive) were dominated in all coupons for all months.

Beech wood Fagus sylvatica dilute-acid hydrolysate as a feedstock to support Chlorella sorokiniana biomass, fatty acid and pigment production.

This work evaluates the possibility of using beech wood (Fagus sylvatica) dilute-acid (H2SO4) hydrolysate as a feedstock for Chlorella sorokiniana growth, fatty acid and pigment production. Neutralized wood acid hydrolysate, containing organic and mineral compounds, was tested on Chlorella growth at different concentrations and compared to growth under phototrophic conditions. Chlorella growth was improved at lower loadings and inhibited at higher loadings. Based on these results, a 12% neutralized wood acid hydrolysate (Hyd12%) loading was selected to investigate its impact on Chlorella growth, fatty acid and pigment production. Hyd12% improved microalgal biomass, fatty acid and pigment productivities both in light and in dark, when compared to photoautotrophic control. Light intensity had substantial influence on fatty acid and pigment composition in Chlorella culture during Hyd12%-based growth. Moreover, heterotrophic Chlorella cultivation with Hyd12% also showed that wood hydrolysate can constitute an attractive feedstock for microalgae cultivation in case of lack of light.

Heterotrophic cultivation of Chlorella pyrenoidosa using sucrose as the sole carbon source by co-culture with Rhodotorula glutinis.

Heterotrophic cultivation of microalgae is a feasible alternative strategy to avoid the light limitation of photoautotrophic culture, but the heterotrophic utilization of disaccharides is difficult for microalgae. Aimed at this problem, a co-culture system was developed by mix culture of C. pyrenoidosa and R. glutinis using sucrose as the sole carbon source. In this system, C. pyrenoidosa could utilize glucose and fructose which were hydrolyzed from sucrose by R. glutinis. The highest specific growth rate and final cell number proportion of algae was 1.02day(-1) and 45%, respectively, when cultured at the initial algal cell number proportion of 95.24% and the final algal cell density was 111.48×10(6)cells/mL. In addition, the lipid content was also promoted due to the synergistic effects in mix culture. This study provides a novel approach using sucrose-riched wastes for the heterotrophic culture of microalgae and may effectively decrease the cost of carbon source.

Production of biomass and lipids by the oleaginous microalgae Monoraphidium sp. QLY-1 through heterotrophic cultivation and photo-chemical modulator induction.

A two-step strategy comprising heterotrophic cultivation and photo-chemical modulator induction was developed to enhance biomass and lipid accumulation in the oleaginous Monoraphidium sp. QLY-1, which was isolated from Qilu Lake in Yunnan Plateau. The algae were first cultivated heterotrophically to achieve high biomass concentration (5.54gL(-1)) with a lipid content of 22.47%. The cultivated algae were diluted, transferred to light environment, and treated with different chemical elicitors. Results showed that the lipid content increased to 36.68% after 3-day of photoinduction. The lipid content was further enhanced by 1.21, 1.32, and 1.29 folds in algal cells treated with nitrogen deficiency, 20gL(-1) NaCl, and 5mM glycine betaine, respectively. The maximum lipid content (48.54%) and lipid productivity (121.27mgL(-1)d(-1)) were obtained in treatments with 20gL(-1) NaCl and 5mM GB, respectively. This study proposes a strategy to efficiently produce lipids by using microalgae.

Lipase-catalyzed in-situ biosynthesis of glycerol-free biodiesel from heterotrophic microalgae, Aurantiochytrium sp. KRS101 biomass.

Heterotrophic microalgae, Aurantiochytrium sp. KRS101 had a large amount of lipid (56.8% total lipids). The cells in the culture medium were easily ruptured due to thin cell wall of Aurantiochytrium sp., which facilitated in-situ fatty acid methyl esters (FAMEs) production directly from biomass. The harvested biomass had a high content of free fatty acids (FFAs), which was advantageous for glycerol-free FAMEs production. FAMEs were directly produced from Aurantiochytrium sp. KRS101 biomass (48.4% saponifiable lipids) using Novozyme 435-catalyzed in-situ esterification in dimethyl carbonate (DMC). DMC was used as a lipid extraction reagent, acyl acceptor and reaction medium. A 433.09mg FAMEs/g biomass was obtained with 89.5% conversion under the optimal condition: DMC to biomass ratio of 5:1 (v/w) and enzyme to biomass ratio of 30% (w/w) at 50°C for 12h. Glycerol could not be detected in the produced FAMEs.

Effects of carbon source and light intensity on the growth and total lipid production of three microalgae under different culture conditions.

We attempted to enhance the growth and total lipid production of three microalgal species, Isochrysis galbana LB987, Nannochloropsis oculata CCAP849/1, and Dunaliella salina, which are capable of accumulating high content of lipid in cells. Low nitrogen concentration under photoautotrophic conditions stimulated total lipid production, but a decreasing total lipid content and an increasing biomass were observed with increasing nitrogen concentration. Among the different carbon sources tested for heterotrophic cultivation, glucose improved the growth of all three strains. The optimal glucose concentration for growth of I. galbana LB987 and N. oculata CCAP849/1 was 0.02 M, and that of D. salina was 0.05 M. Enhanced growth occurred when they were cultivated under heterotrophic or mixotrophic conditions compared with photoautotrophic conditions. Meanwhile, high total lipid accumulation in cells occurred when they were cultivated under photoautotrophic or mixotrophic conditions. During mixotrophic cultivation, biomass production was not affected significantly by light intensity; however, both chlorophyll concentration and total lipid content increased dramatically with increasing light intensity up to 150 µmol/m(2)/s. The amount and composition ratio of saturated and unsaturated fatty acids in cells were different from each other depending on both species and light intensity. The highest accumulation of total fatty acid (C16-C18) among the three strains was found from cells of N. oculata CCAP849/1, which indicates that this species can be used as a source for production of biodiesel.

Carbon-to-nitrogen ratio affects the biomass composition and the fatty acid profile of heterotrophically grown Chlorella sp. TISTR 8990 for biodiesel production.

Chlorella sp. TISTR 8990 was cultivated heterotrophically in media with various initial carbon-to-nitrogen ratios (C/N ratio) and at different agitation speeds. The production of the biomass, its total fatty acid content and the composition of the fatty acids were affected by the C/N ratio, but not by agitation speed in the range examined. The biomass production was maximized at a C/N mass ratio of 29:1. At this C/N ratio, the biomass productivity was 0.68gL(-1)d(-1), or nearly 1.6-fold the best attainable productivity in photoautotrophic growth. The biomass yield coefficient on glucose was 0.62gg(-1) during exponential growth. The total fatty acids (TFAs) in the freeze-dried biomass were maximum (459mgg(-1)) at a C/N ratio of 95:1. Lower values of the C/N ratio reduced the fatty acid content of the biomass. The maximum productivity of TFAs (186mgL(-1)d(-1)) occurred at C/N ratios of 63:1 and higher. At these conditions, the fatty acids were mostly of the polyunsaturated type. Allowing the alga to remain in the stationary phase for a prolonged period after N-depletion, reduced the level of monounsaturated fatty acids and the level of polyunsaturated fatty acids increased. Biotin supplementation of the culture medium reduced the biomass productivity relative to biotin-free control, but had no effect on the total fatty acid content of the biomass.

Impacts of Silver Nanoparticles on a Natural Estuarine Plankton Community.

Potential effects of metal nanoparticles on aquatic organisms and food webs are hard to predict from the results of single-species tests under controlled laboratory conditions, and more realistic exposure experiments are rarely conducted. We tested whether silver nanoparticles (Ag NPs) had an impact on zooplankton grazing on their prey, specifically phytoplankton and bacterioplankton populations. If Ag NPs directly reduced the abundance of prey, thereby causing the overall rate of grazing by their predators to decrease, a cascading effect on a planktonic estuarine food web would be seen. Our results show that the growth rates of both phytoplankton and bacterioplankton populations were significantly reduced by Ag NPs at concentrations of ≥500 µg L(-1). At the same time, grazing rates on these populations tended to decline with exposure to Ag NPs. Therefore, Ag NPs did not cause a cascade of effects through the food web but impacted a specific trophic level. Photosynthetic efficiency of the phytoplankton was significantly reduced at Ag NPs concentrations of ≥500 µg L(-1). These effects did not occur at relatively low concentrations of Ag that are often toxic to single species of bacteria and other organisms, suggesting that the impacts of Ag NP exposure may not be apparent at environmentally relevant concentrations due to compensatory processes at the community level.

Quaternary ammonium polyethylenimine nanoparticles for treating bacterial contaminated water.

This study highlights the potential application of antimicrobial quaternary ammonium nanomaterials for water disinfection. Quaternary ammonium polyethylenimine (QA-PEI) nanoparticles (NPs) were synthesized by polyethylenimine crosslinking and alkylation with octyl iodide followed by methyl iodide quaternization. Particles modified with octyldodecyl alkyl chains were also prepared and evaluated. The antimicrobial activity of QA-PEI NPs was studied after anchoring in non-leaching polymeric coatings and also in aqueous suspension. Particles at different loadings (w/w) were embedded in polyethylene vinyl acetate and polyethylene methacrylic acid coatings and tested for antimicrobial activity against four representative strains of bacteria in static and dynamic modes. Coatings embedded with fluorescent labelled particles tracked by Axioscope fluorescence microscope during the antimicrobial test indicates no particles leaching out. Coatings loaded with 5% w/w QA-PEI exhibited strong antibacterial activity. Aqueous suspension was tested and found effective for bacterial decontamination at 0.1 ppm and maintains its activity for several weeks.

Prospective technology on bioethanol production from photofermentation.

The most important global demand is the energy supply from alternative source. Ethanol may be considered an environmental friendly fuel that has been produced by feedstock. The production of ethanol by microalgae represent a process with reduced environmental impact with efficient CO2 fixation and requiring less arable land. This work studied the production of ethanol from green alga Chlamydomonas reinhardtii through the cellular metabolism in a light/dark cycle at 25 °C in a TAP medium with sulfur depletion. The parameters evaluated were inoculum concentration and the medium supplementation with mixotrophic carbon sources. The combination of C.reinhardtii and Rhodobacter capsulatus through a hybrid or co-culture systems was also investigated as well. C.reinhardtii maintained in TAP-S produced 19.25±4.16 g/L (ethanol). In addition, in a hybrid system, with medium initially supplemented with milk whey permeated and the algal effluent used by R. capsulatus, the ethanol production achieved 19.94±2.67 g/L.

Bioaugmentation treatment of municipal wastewater with heterotrophic-aerobic nitrogen removal bacteria in a pilot-scale SBR.

PCN bacteria capable of heterotrophic-aerobic nitrogen removal was successfully applied for bioaugmented treatment of municipal wastewater in a pilot-scale SBR. At an appropriate COD/N ratio of 8, the bioaugmentation system exhibited stable and excellent carbon and nutrients removal, the averaged effluent concentrations of COD, NH4(+)-N, TN and TP were 20.6, 0.69, 14.1 and 0.40 mg/L, respectively, which could meet the first class requirement of the National Municipal Wastewater Discharge Standards of China (COD<50 mg/L, TN<15 mg/L, TP<0.5 mg/L). Clone library and real-time PCR analysis revealed that the introduced bacteria greatly improved the structure of original microbial community and facilitated their aerobic nutrients removal capacities. The proposed emerging technology was shown to be an alternative technology to establish new wastewater treatment systems and upgrade or retrofit conventional systems from secondary-level to tertiary-level.