Responses of Chlamydomonas reinhardtii during the transition from P-deficient to P-sufficient growth (the P-overplus response): The roles of the vacuolar transport chaperones and polyphosphate synthesis.

Phosphorus (P) assimilation and polyphosphate (polyP) synthesis were investigated in Chlamydomonas reinhardtii by supplying phosphate (PO43- ; 10 mg P·L-1 ) to P-depleted cultures of wildtypes, mutants with defects in genes involved in the vacuolar transporter chaperone (VTC) complex, and VTC-complemented strains. Wildtype C. reinhardtii assimilated PO43- and stored polyP within minutes of adding PO43- to cultures that were P-deprived, demonstrating that these cells were metabolically primed to assimilate and store PO43- . In contrast, vtc1 and vtc4 mutant lines assayed under the same conditions never accumulated polyP, and PO43- assimilation was considerably decreased in comparison with the wildtypes. In addition, to confirm the bioinformatics inferences and previous experimental work that the VTC complex of C. reinhardtii has a polyP polymerase function, these results evidence the influence of polyP synthesis on PO43- assimilation in C. reinhardtii. RNA-sequencing was carried out on C. reinhardtii cells that were either P-depleted (control) or supplied with PO43- following P depletion (treatment) in order to identify changes in the levels of mRNAs correlated with the P status of the cells. This analysis showed that the levels of VTC1 and VTC4 transcripts were strongly reduced at 5 and 24 h after the addition of PO43- to the cells, although polyP granules were continuously synthesized during this 24 h period. These results suggest that the VTC complex remains active for at least 24 h after supplying the cells with PO43- . Further bioassays and sequence analyses suggest that inositol phosphates may control polyP synthesis via binding to the VTC SPX domain.

Nitrous oxide emissions during microalgae-based wastewater treatment: current state of the art and implication for greenhouse gases budgeting.

Microalgae can synthesise the ozone depleting pollutant and greenhouse gas nitrous oxide (N2O). Consequently, significant N2O emissions have been recorded during real wastewater treatment in high rate algal ponds (HRAPs). While data scarcity and variability prevent meaningful assessment, the magnitude reported (0.13-0.57% of the influent nitrogen load) is within the range reported by the Intergovernmental Panel on Climate Change (IPCC) for direct N2O emissions during centralised aerobic wastewater treatment (0.016-4.5% of the influent nitrogen load). Critically, the ability of microalgae to synthesise N2O challenges the IPCC's broad view that bacterial denitrification and nitrification are the only major cause of N2O emissions from wastewater plants and aquatic environments receiving nitrogen from wastewater effluents. Significant N2O emissions have indeed been repeatedly detected from eutrophic water bodies and wastewater discharge contributes to eutrophication via the release of nitrogen and phosphorus. Considering the complex interplays between nitrogen and phosphorus supply, microalgal growth, and microalgal N2O synthesis, further research must urgently seek to better quantify N2O emissions from microalgae-based wastewater systems and eutrophic ecosystems receiving wastewater. This future research will ultimately improve the prediction of N2O emissions from wastewater treatment in national inventories and may therefore affect the prioritisation of mitigation strategies.

Escherichia coli removal during domestic wastewater treatment in outdoor high rate algae ponds: long-term performance and mechanistic implications.

Escherichia coli (E. coli) first-order decay rates ranging from 3.34 to 11.9 d-1 (25-75% data range, N = 128) were recorded in two outdoor pilot-scale (0.88 m3) high rate algal ponds (HRAPs) continuously fed primary domestic wastewater over two years (influent E. coli cell count of 4.74·106 ± 3.37·106 MPN·100 mL-1, N = 142). The resulting removal performance was relatively constant throughout the year (log10-removal averaging 1.77 ± 0.54, N = 128), apart from a significant performance drop during a cold rainy period. E. coli removal performance was not strongly correlated to any of the meteorological or operational parameters recorded (e.g. sunlight intensity, pH, temperature). Hourly monitoring of E. coli cell count evidenced that E. coli removal, pH, dissolved oxygen (DO) and pond temperature peaked in the late afternoon of sunny summer days. Such improved daytime removal was, however, not evidenced in spring, even under sunny conditions causing milder increases in pH, DO and temperature. Overall, the data confirm the potential of HRAPs to support efficient E. coli removal during secondary domestic wastewater treatment and suggests E. coli decay was mainly caused by dark mechanisms episodically enhanced by indirect sunlight-mediated mechanisms and/or high pH toxicity.

The biosynthesis of nitrous oxide in the green alga Chlamydomonas reinhardtii.

Over the last decades, several studies have reported emissions of nitrous oxide (N2 O) from microalgal cultures and aquatic ecosystems characterized by a high level of algal activity (e.g. eutrophic lakes). As N2 O is a potent greenhouse gas and an ozone-depleting pollutant, these findings suggest that large-scale cultivation of microalgae (and possibly, natural eutrophic ecosystems) could have a significant environmental impact. Using the model unicellular microalga Chlamydomonas reinhardtii, this study was conducted to investigate the molecular basis of microalgal N2 O synthesis. We report that C. reinhardtii supplied with nitrite (NO2- ) under aerobic conditions can reduce NO2- into nitric oxide (NO) using either a mitochondrial cytochrome c oxidase (COX) or a dual enzymatic system of nitrate reductase (NR) and amidoxime-reducing component, and that NO is subsequently reduced into N2 O by the enzyme NO reductase (NOR). Based on experimental evidence and published literature, we hypothesize that when nitrate (NO3- ) is the main Nitrogen source and the intracellular concentration of NO2- is low (i.e. under physiological conditions), microalgal N2 O synthesis involves the reduction of NO3- to NO2- by NR followed by the reduction of NO2- to NO by the dual system involving NR. This microalgal N2 O pathway has broad implications for environmental science and algal biology because the pathway of NO3- assimilation is conserved among microalgae, and because its regulation may involve NO.

Fast algal eco-toxicity assessment: Influence of light intensity and exposure time on Chlorella vulgaris inhibition by atrazine and DCMU.

In order to develop a rapid assay suitable for algal eco-toxicity assessments under conditions representative of natural ecosystems, this study evaluated the short-term (<1h) response of algae exposed to atrazine and DCMU using oxygen productivity measurements. When Chlorella vulgaris was exposed to these herbicides under 'standard' low light intensity (as prescribed by OECD201 guideline), the 20min-EC50 values recorded via oxygen productivity (atrazine: 1.32±0.07µM; DCMU: 0.31±0.005µM) were similar the 96-h EC50 recorded via algal growth (atrazine: 0.56µM; DCMU: 0.41µM), and within the range of values reported in the literature. 20min-EC50 values increased by factors of 3.0 and 2.1 for atrazine and DCMU, respectively, when light intensity increased from 60 to 1400µmolm-2s-1 of photosynthetically active radiation, or PAR. Further investigation showed that exposure time significantly also impacted the sensitivity of C. vulgaris under high light intensity (>840µmolm-2s-1 as PAR) as the EC50 for atrazine and DCMU decreased by up to 6.2 and 2.1 folds, respectively, after 50min of exposure at a light irradiance of 1400µmolm-2s-1 as PAR. This decrease was particularly marked at high light intensities and low algae concentrations and is explained by the herbicide disruption of the electron transfer chain triggering photo-inhibition at high light intensities. Eco-toxicity assessments aiming to understand the potential impact of toxic compounds on natural ecosystems should therefore be performed over sufficient exposure times (>20min for C. vulgaris) and under light intensities relevant to these ecosystems.

Maximizing Productivity and Reducing Environmental Impacts of Full-Scale Algal Production through Optimization of Open Pond Depth and Hydraulic Retention Time.

The ability to dynamically control algal raceway ponds to maximize biomass productivity and reduce environmental impacts (e.g., land and water use) with consideration of local constraints (e.g., water availability and climatic conditions) is an important consideration in algal biotechnology. This paper presents a novel optimization strategy that seeks to maximize growth (i.e., optimize land use), minimize respiration losses, and minimize water demand through regular adjustment of pond depth and hydraulic retention time (HRT) in response to seasonal changes. To evaluate the efficiency of this strategy, algal productivity and water demand were simulated in five different climatic regions. In comparison to the standard approach (constant and location-independent depth and HRT), dynamic control of depth and HRT was shown to increase productivity by 0.6-9.9% while decreasing water demand by 10-61% depending upon the location considered (corresponding to a decrease in the water footprint of 19-62%). Interestingly, when the fact that the water demand was limited to twice the local annual rainfall was added as a constraint, higher net productivities were predicted in temperate and tropical climates (15.7 and 16.7 g m(-2) day(-1), respectively) than in Mediterranean and subtropical climates (13.0 and 9.7 g m(-2) day(-1), respectively), while algal cultivation was not economically feasible in arid climates. Using dynamic control for a full-scale operation by adjusting for local climatic conditions and water constraints can notably affect algal productivity. It is clear that future assessments of algal cultivation feasibility should implement locally optimized dynamic process control.

Algal productivity modeling: a step toward accurate assessments of full-scale algal cultivation.

A new biomass productivity model was parameterized for Chlorella vulgaris using short-term (<30 min) oxygen productivities from algal microcosms exposed to 6 light intensities (20-420 W/m(2)) and 6 temperatures (5-42 °C). The model was then validated against experimental biomass productivities recorded in bench-scale photobioreactors operated under 4 light intensities (30.6-74.3 W/m(2)) and 4 temperatures (10-30 °C), yielding an accuracy of ± 15% over 163 days of cultivation. This modeling approach addresses major challenges associated with the accurate prediction of algal productivity at full-scale. Firstly, while most prior modeling approaches have only considered the impact of light intensity on algal productivity, the model herein validated also accounts for the critical impact of temperature. Secondly, this study validates a theoretical approach to convert short-term oxygen productivities into long-term biomass productivities. Thirdly, the experimental methodology used has the practical advantage of only requiring one day of experimental work for complete model parameterization. The validation of this new modeling approach is therefore an important step for refining feasibility assessments of algae biotechnologies.

Full-scale validation of a model of algal productivity.

While modeling algal productivity outdoors is crucial to assess the economic and environmental performance of full-scale cultivation, most of the models hitherto developed for this purpose have not been validated under fully relevant conditions, especially with regard to temperature variations. The objective of this study was to independently validate a model of algal biomass productivity accounting for both light and temperature and constructed using parameters experimentally derived using short-term indoor experiments. To do this, the accuracy of a model developed for Chlorella vulgaris was assessed against data collected from photobioreactors operated outdoor (New Zealand) over different seasons, years, and operating conditions (temperature-control/no temperature-control, batch, and fed-batch regimes). The model accurately predicted experimental productivities under all conditions tested, yielding an overall accuracy of ±8.4% over 148 days of cultivation. For the purpose of assessing the feasibility of full-scale algal cultivation, the use of the productivity model was therefore shown to markedly reduce uncertainty in cost of biofuel production while also eliminating uncertainties in water demand, a critical element of environmental impact assessments. Simulations at five climatic locations demonstrated that temperature-control in outdoor photobioreactors would require tremendous amounts of energy without considerable increase of algal biomass. Prior assessments neglecting the impact of temperature variations on algal productivity in photobioreactors may therefore be erroneous.

Evaluation of Extraction Techniques for Recovery of Microalgal Lipids under Different Growth Conditions.

Microalgal lipids contain a wide array of liposoluble bioactive compounds, but lipid extraction remains a critical limitation for their commercial use. An accelerated solvent extraction (ASE) was used to extract lipids from Chlamydomonas reinhardtii, Arthrospira platensis (Spirulina), and Chlorella vulgaris grown under either standard or nitrogen depletion conditions. Under standard growth conditions, ASE using methanol:chloroform (2:1), methyl tert-butyl ether (MTBE):methanol:water, and ethanol at 100 °C resulted in the highest recovery of total lipids (352 ± 30, 410 ± 32, and 127 ± 15 mg/g biomass from C. reinhardtii, C. vulgaris, and A. platensis, respectively). Similarly, the highest total lipid and triacylglycerols (TAGs) recovery from biomass cultivated under nitrogen depletion conditions was found at 100 °C using methanol:chloroform, for C. reinhardtii (total, 550 ± 21; TAG, 205 ± 2 mg/g biomass) and for C. vulgaris (total, 612 ± 29 mg/g; TAG, 253 ± 7 mg/g biomass). ASE with MTBE:methanol:water at 100 °C yielded similar TAG recovery for C. reinhardtii (159 ± 6 mg/g) and C. vulgaris (200 ± 4 mg/g). Thus, MTBE:methanol:water is suggested as an alternative substitute to replace hazardous solvent mixtures for TAGs extraction with a much lower environmental impact. The extracted microalgal TAGs were rich in palmitic (C16:0), stearic (C18:0), oleic (C18:1,9), linoleic (C18:2n6), and α-linolenic (C18:3n3) acids. Under nitrogen depletion conditions, increased palmitic acid (C16:0) recovery up to 2-fold was recorded from the biomasses of C. reinhardtii and C. vulgaris. This study demonstrates a clear linkage between the extraction conditions applied and total lipid and TAG recovery.

Outdoor cultivation of temperature-tolerant Chlorella sorokiniana in a column photobioreactor under low power-input.

Temperature-tolerant Chlorella sorokiniana was cultivated in a 51-L column photobioreactor with a 1.1 m(2) illuminated area. The reactor was operated outdoors under tropical meteorological conditions (Singapore) without controlling temperature and the culture was mixed at a power input of 7.5 W/m(3) by sparging CO(2) -enriched air at 1.2 L/min (gas hold-up of 0.02). Biomass productivity averaged 10 ± 2.2 g/m(2) -day over six batch studies, yielding an average photosynthetic efficiency (PE) of 4.8 ± 0.5% of the total solar radiation (P = 0.05, N = 6). This demonstrates that temperature-tolerant microalgae can be cultivated at high PE under a mixing input sevenfold to ninefold lower than current operational guidelines (50-70 W/m(3)) and without the need for temperature control (the culture broth temperature reached 41 °C during operation). In this study, the PE value was determined based on the amount of solar radiation actually reaching the algae and this amount was estimated using a mathematical model fed with onsite solar irradiance data. This determination was found to be particularly sensitive to the value of the atmospheric diffusion coefficient, which generated a significant uncertainty in the PE calculation. The use of the mathematical model, however, confirmed that the vertical reactor geometry supported efficient photosynthesis by reducing the duration and intensity of photoinhibition events. The model also revealed that all three components of direct, diffuse, and reflected solar radiation were quantitatively important for the vertical column photobioreactor, accounting for 14%, 65%, and 21% of the total solar radiation reaching the culture, respectively. The accurate prediction of the discrete components of solar radiation reaching the algae as a function of climatic, geographic, and design parameters is therefore crucial to optimize the individual reactor geometry and the layout/spacing between the individual reactors in a reactor farm.

Questioning the accuracy of greenhouse gas accounting from agricultural waste: a case study.

The New Zealand Greenhouse Gas Inventory (the NZ Inventory) uses country-specific data to quantify CH emissions from anaerobic ponds treating dairy farm effluent (315 Gg CO equivalent [CO-e] in 2009). In this study, we used literature data to: (i) evaluate the accuracy of the NZ Inventory's parameters used to quantify these CH emissions; and (ii) determine whether the NZ Inventory's scope is capturing the full spectrum of sources with bio-CH potential entering anaerobic ponds. The research indicated that the current NZ Inventory methodology is underestimating CH emissions from anaerobic ponds across New Zealand by 264 to 603 Gg CO-e annually. Moreover, the NZ Inventory is currently not accounting for (i) manure from supplementary feed pads and stand-off pads (annual CH emissions = 207-330 Gg CO-e); (ii) waste milk (153-280 Gg CO-e); and (iii) supplementary feed waste (90-216 Gg CO-e). Annual CH emissions from anaerobic ponds on dairy farms across New Zealand are thus more likely to be 1029 to 1744 Gg CO-e, indicating that the NZ Inventory is reporting as little as 18% of actual CH emissions produced by this sector. These additional wastes are not accounted for in the methodology prescribed by the Intergovernmental Panel on Climate Change for estimating CH emissions from dairy manure. Consequently, other significant dairying nations will also probably be underestimating their waste CH emissions. Our research highlights that, if governments attempt to include country-specific emission factors in their greenhouse gas inventories, these factors must be based on an assessment of the full spectrum of sources contributing to greenhouse gas emissions within any given sector.

Study from microcosms and mesocosms reveals Escherichia coli removal in high rate algae ponds during domestic wastewater treatment is primarily caused by dark decay.

While high rate algal ponds (HRAPs) can provide efficient pathogen removal from wastewater, the mechanisms involved remain unclear. To address this knowledge gap, the mechanisms potentially causing Escherichia coli (E. coli) removal during microalgae-based wastewater treatment were successively assessed using laboratory microcosms designed to isolate known mechanisms, and bench scale assays performed in real HRAP broth. During laboratory assays, E. coli decay was only significantly increased by alkaline pH (above temperature-dependent thresholds) due to pH induced toxicity, and direct sunlight exposure via UV-B damage and/or endogenous photo-oxidation. Bench assays confirmed alkaline pH toxicity caused significant decay but sunlight-mediated decay was not significant, likely due to light attenuation in the HRAP broth. Bench assays also evidenced the existence of uncharacterized 'dark' decay mechanism(s) not observed in laboratory microcosms. To numerically evaluate the contribution of each mechanism and the uncertainty associated, E. coli decay was modelled assuming dark decay, alkaline pH induced toxicity, and direct sunlight-mediated decay were independent mechanisms. The simulations confirmed E. coli decay was mainly caused by dark decay during bench assays (48.2-89.5% estimated contribution to overall decay at the 95% confidence level), followed by alkaline-pH induced toxicity (8.3-46.5%), and sunlight-mediated decay (0.0-21.9%).

Universal temperature model for shallow algal ponds provides improved accuracy.

While temperature is fundamental to the design and optimal operation of shallow algal ponds, there is currently no temperature model universally applicable to these systems. This paper presents a model valid for any opaque water body of uniform temperature profile. This new universal model was tested against 1 year of experimental data collected from a wastewater treatment high rate algal pond. On the basis of 1 year of data collected every 15 min, the average errors of the predicted afternoon peak and predawn minimum were both only 1.3 °C and the average error between these extremes was just 1.2 °C. In order to demonstrate the improvement in accuracy gained, the expressions for heat fluxes used in nine prior temperature models were systematically substituted into the new universal model and evaluated against the experimental data. Errors in the peak and minimum temperatures increased by up to 2.1 and 3.2 °C, respectively, while the error between these extremes increased by up to 2.9 °C. In practical applications, these levels of inaccuracies could lead to an under/overestimation of the algal productivity and the evaporative water loss by approximately 40% and 300%, respectively.

Overcoming substrate inhibition during biological treatment of monoaromatics: recent advances in bioprocess design.

The biological removal of monoaromatic compounds from contaminated environments, usually arising from industrial activity, is challenging because of the inherent toxicity of these compounds to microorganisms, particularly at the concentrations that can be encountered in industrial waste streams. A wide range of bioprocess designs have been proposed and tested with the aim of achieving high removal efficiencies, with varying degrees of technical success, and potential for practical implementation. This review reports on the progress on variations of well-known themes made in the last 3-4 years, as well as new bioprocess technologies that address the cytotoxicity of monoaromatics directly. Areas for further research are also proposed.

Chlamydomonas reinhardtii, an Algal Model in the Nitrogen Cycle.

Nitrogen (N) is an essential constituent of all living organisms and the main limiting macronutrient. Even when dinitrogen gas is the most abundant form of N, it can only be used by fixing bacteria but is inaccessible to most organisms, algae among them. Algae preferentially use ammonium (NH4+) and nitrate (NO3-) for growth, and the reactions for their conversion into amino acids (N assimilation) constitute an important part of the nitrogen cycle by primary producers. Recently, it was claimed that algae are also involved in denitrification, because of the production of nitric oxide (NO), a signal molecule, which is also a substrate of NO reductases to produce nitrous oxide (N2O), a potent greenhouse gas. This review is focused on the microalga Chlamydomonas reinhardtii as an algal model and its participation in different reactions of the N cycle. Emphasis will be paid to new actors, such as putative genes involved in NO and N2O production and their occurrence in other algae genomes. Furthermore, algae/bacteria mutualism will be considered in terms of expanding the N cycle to ammonification and N fixation, which are based on the exchange of carbon and nitrogen between the two organisms.

Biofilm photobioreactors for the treatment of industrial wastewaters.

A flat plate and a tubular packed-bed photobioreactor with an algal-bacterial biofilm attached onto Poraver beads carriers, a flat plate and a tubular photobioreactor with the biofilm attached onto the reactor walls, and an algal-turf reactor were compared in terms of BOD removal efficiencies, elimination capacities, and stability. A control column photobioreactor with suspended algal-bacterial biomass was also tested to compare the performance of biofilm photobioreactors with conventional algal-based processes. When the algal-bacterial biomass was immobilized onto Poraver the process never reached a steady state due to a poor homogenization in the bioreactor. When the biofilm was formed onto the reactor wall (or reactor base) the process was stable. A maximum degradation rate of 295mg BODl(-1)h(-1) was achieved in the algal-turf reactor although control experiments performed in the dark showed atmospheric O2 diffusion represented 55% of the oxygenation capacity in this system. BOD removal rates of 108, and 92mg BODl(-1)h(-1) were achieved in the tubular and flat plate biofilm reactors, respectively, compared to 77mg BODl(-1)h(-1) in the control suspended bioreactor. In addition, all biofilm photobioreactors produced an easily settleable biomass. Evidence was found that biomass attachment to the reactor's wall improved stability.

Biological treatment of indoor air for VOC removal: potential and challenges.

There is nowadays no single fully satisfactory method for VOC removal from indoor air due to the difficulties linked to the very low concentration (microg m(-3) range), diversity, and variability at which VOCs are typically found in the indoor environment. Although biological methods have shown a certain potential for this purpose, the specific characteristic of indoor air and the indoor air environment brings numerous challenges. In particular, new methods must be developed to inoculate, express, and maintain a suitable and diverse catabolic ability under conditions of trace substrate concentration which might not sustain microbial growth. In addition, the biological treatment of indoor air must be able to purify large amounts of air in confined environments with minimal nuisances and release of microorganisms. This requires technical innovations, the development of specific testing protocols and a deep understanding of microbial activities and the mechanisms of substrate uptake at trace concentrations.

Two-phase partitioning bioreactor for the biodegradation of high concentrations of pentachlorophenol using Sphingobium chlorophenolicum DSM 8671.

An organic-aqueous two-liquid-phase partitioning system was developed to degrade high concentrations of pentachlorophenol (PCP). Dioctyl sebacate was selected among 12 non-aqueous phases as the most suitable solvent to control the delivery of PCP to the aqueous phase for being non-biodegradable and biocompatible. In shake-flask experiments, the two-phase system was able to support the removal of 1g PCP l(-1) of total liquid phase. The performance of the two-liquid phase partitioning system (TLPPS) in shake-flask was evaluated under different conditions. At the initial biomass concentrations of 7, 25, and 58 mg dry weight l(-1), the volumetric removal rates of PCP obtained were 25.7+/-0.5, 32.1+/-0.1, and 39.3+/-2.9 mg PCP l(-1)h(-1), respectively. Higher performance was observed at lower organic-aqueous phase ratios (16% and 28%) than higher ones (37% and 44%). In a 2-l TLPPS, the degradation of 10 g PCP was completed in less than 100 h at a total volumetric rate of 142 mg l(-1) h(-1). Kinetics study using Monod model showed that compared to monophasic systems, the biphasic system significantly enhanced the maximum specific growth rate and PCP removal rate. Results of this biphasic system showed no accumulation of unknown by-product(s) which has been reported for physical-pretreatment or high-performance biphasic systems of PCP degradation.

Continuous biodegradation of 17beta-estradiol and 17alpha-ethynylestradiol by Trametes versicolor.

The feasibility of 17beta-estradiol (E2) and 17alpha-ethynylestradiol (EE2) removal by Trametes versicolor was demonstrated in batch and continuous cultures. In batch, E2 and EE2 initially supplied at 10 mg l(-1) were removed by more than 97% in 24h, which corresponded to volumetric removal rates of 0.43 and 0.44 mg l(-1)h(-1), respectively. A bioreactor inoculated with T. versicolor pellets was then continuously operated during 26 days at a hydraulic retention time of 120 h. E2 and EE2 were completely removed at volumetric removal rates of 0.16 and 0.09 mg l(-1)h(-1), respectively, when fed at 18.8 and 7.3 mg l(-1), respectively. Evidence was found that removal was caused by laccase. This study demonstrates the technical feasibility of fungal treatment of estrogens using continuous bioreactor with suspended fungal biomass.