Iron salt-based chemically enhanced primary treatment effectively captures organic carbon from municipal wastewater, providing a concentrated substrate for methane recovery through anaerobic digestion. However, methane production from iron salt-based chemically enhanced primary sedimentation (Fe-CEPS) sludge remains substantially lower than expected, and the mechanisms responsible for this reduction remain poorly understood. Here, the relationship between iron-bound organic carbon (Fe-OC) formation and methane production was investigated by combining anaerobic digestion experiments with iron mineral-glucose model systems. Increasing Fe dosage reduced methane yield from 221.1 to 74.7 mL CH4 g-1 VS while promoting Fe-OC accumulation. A strong negative correlation was observed between Fe-OC content and methane production. Fresh Fe-CEPS sludge contained only limited Fe-OC (<10% of total organic carbon), whereas Fe-OC accounted for approximately 26% of total organic carbon after anaerobic digestion, indicating that Fe-OC formed predominantly during digestion rather than during coagulation. Iron speciation analyses revealed extensive Fe(III) reduction accompanied by secondary iron mineral formation. Model experiments further demonstrated that Fe(II)-induced mineral transformation increased mineral crystallinity, enhanced organic carbon stabilization, reduced carbon bioavailability, and consequently suppressed methane production. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy further revealed progressively stronger spatial associations between organic carbon and iron minerals as mineral crystallinity increased. These findings reveal a previously unrecognized mechanism whereby Fe(II)-induced secondary iron mineral formation promotes Fe-OC accumulation and reduces organic carbon bioavailability during anaerobic digestion. Regulating iron mineral transformation may therefore represent an effective strategy for improving methane recovery from iron-enhanced primary sludge.

Sulfate dynamics strongly regulate methane emissions in eutrophic freshwater lakes, yet the mechanisms driving in situ sulfate regeneration remain poorly understood. This study examined the temporal and spatial variations of sulfate and sulfur-cycling microorganisms in Lake Taihu to elucidate the influence of dissolved oxygen (DO) on sulfate regeneration and methane fluxes. Pronounced seasonal sulfate fluctuations were observed with higher concentrations during oxic (spring and winter) and lower levels during anoxic (summer and autumn) periods. Under oxic conditions, sulfur-oxidizing bacteria (SOB) dominated surface sediments, whereas sulfate-reducing bacteria (SRB) prevailed in deeper anoxic layers, sustaining an active internal sulfur cycle. Microcosm experiments demonstrated that sulfate was regenerated from reduced sulfur species under oxic conditions, and inhibition of sulfate reduction by molybdate confirmed the oxidative recycling process. Increased sulfate availability substantially suppressed methane emissions by outcompeting methanogens for organic substrates. Quantitative analysis indicated that approximately 43% of the methane reduction was attributable to the regeneration of sulfate and other electron acceptors (e.g., nitrate and Fe(III)), while 57% was due to aerobic methane oxidation. These findings highlight a redox-driven "sulfur pump", "iron pump", and "nitrogen pump" mechanism that collectively mitigates methane emissions, offering new insights into biogeochemical feedbacks in shallow lake ecosystems.

The microalgal research field is currently lacking a unified theoretical computing system to explain various experimental results related to microalgal growth. Thus a novel universal theoretical model was created to predict microalgal growth with carbon dioxide (CO2) fixation in any cultivation system. First, a new "light-effect colorimetric method" was proposed to estimate the actual f(2) value of suspended microalgal cells during regular experimentation using a formula explaining the photosynthetic effective electron transfer rate (ETR = PFD center dot phi II center dot f(1)center dot f(2)), which only requires the use of a simple spectrophotometer. A mathematical relationship between photosynthetic electron transfer and the microalgal growth rate was then identified based on this modified ETR by simplifying factors influencing the cultivation conditions (e.g., nutrients and CO2) into the slope and intercept of this formula. Subsequently, software was written to allow the above relationship to stimulate any 3-D microalgal cultivation system. Many example cases were conducted to clarify the significance and application of this theoretical model. It was found that the average ETR of a cultivation system describes microalgal tolerance to high CO2 concentrations. A photobioreactor at any given location under a certain light condition has a theoretical maximum yield of microalgal biomass, irrespective of how the other cultivation conditions change. A new concept of "biological similarity" is proposed as a basic principle for scaling up microalgal experiments with photosynthetic CO2 fixation to perform a repeated growth curve with < 5 % error. Finally, a "multi-batch dilution method" was demonstrated to increase the microalgal biomass yield by 64.4 % over a short cultivation period. General application of this calculation model would change the empirical status of microalgal engineering designs.

High microalgal growth rates depend on appropriate solution mixing, and therefore, mass transfer can be achieved by installing baffle structures into photobioreactor (PBR) systems. In this study, butterfly-shaped baffles were installed in a double column photobioreactor (DC-PBR) to decrease the mixing time, increase the mass transfer coefficient, and promote the growth rate of Arthrospira platensis. The mixing time was reduced by 20%, increasing the baffle size and the angle between wings, resulting in the mass transfer coefficient being simultaneously enhanced by 32%. The vertical flow vortices generated by the butterfly baffles strengthened the light/dark cycle between the inner and outer columns in DC-PBRs, which improved the chlorophyll-a content by 19%, photochemical efficiency and electron transfer rate by 20% during photosynthesis. The biomass growth rate of A. platensis was increased by 33% with the addition of a butterfly baffle to the PBR, while the helix pitch and trichome length were enlarged by 15-16%.

This study determined that 60% is the most appropriate concentration of CO2 for the domestication of microalgae to obtain strains with improved flue gas CO2-adapting ability. The effect of long-term high CO2 stress (6-99% concentrations) on microalgal gene mutations was first clarified with genomic and transcriptomic analyses. The most beneficial long fragment indel/SV gene mutations in microalgae were obtained under 60% CO2. However, > 60% CO2 domestication caused genotoxicity of the microalgae cells via the following mechanisms: (1) it was not conducive to forming more stable long fragment indel/SV gene mutations, thus preventing further gene mutation; (2) gene mutations did not generate successful linkage to the regulation in transcription and translation; and (3) inhibition of the mismatch repair damaged the specialized ability of genetic variation, leading to the disrepair of harmful gene mismatches and fewer beneficial mutations. These novel results revealed that higher concentrations of CO2 for microalgal domestication did not necessarily result in microalgae that were tolerant to CO2 owing to the genotoxicity of long-term high CO2 stress. This conclusion informs futures efforts of domestication in the microalgae industry.

Microalgae-photosynthetic bacteria (PSB) co-culture, which is promising for wastewater treatment and lipid production, is lacking of study. In this work, the combinations of 3 microalgae and 3 PSB strains were firstly screened and then different inoculation ratios of the co-cultures were investigated. It was found the best promotion was Chlorella pyrenoidosa/Rhodobacter capsulatus co-culture (1:1), where the biomass productivity, acetate assimilation rate and lipid productivity were 1.64, 1.61 and 2.79 times than that of the sum of pure microalgae and PSB cultures, respectively. Meanwhile, the inoculation ratio significantly affected the growth rate and lipid productivity of co-culture systems. iTRAQ analysis showed that PSB played a positive effect on acetate assimilation, TCA cycle and glyoxylate cycle of microalgae, but decreased the carbon dioxide utilization and photosynthesis, indicating PSB promoted the microalgae metabolism of organic carbon utilization and weakened inorganic carbon utilization. These findings provide in-depth understanding of carbon utilization in microalgae-PSB co-culture.

In order to reutilize Fe2O3 particles in flue gas from coal-fired power plant as a ferrum nutrient for improving microalgae growth, Na-Citrate was proposed to chelate FeCl3 derived from Fe2O3 and HCl reactions to promote biomass and lipid productivities of Chlorella PY-ZU1. Fe-Citrate gave much higher biomass and lipid productivities than FeCl3, Fe-EDTA, Fe-DTPA and Fe-HEDTA, because organic chelator prevented Fe3+ from depositing, lower stability constant resulted in easier dissociation of ferric chelate, smaller chelate facilitated Fe2+ (reduced from Fe3+) transportation through cell membranes. The biomass growth and photosynthetic capacity of Chlorella PY-ZU1 cultivated with Fe-Citrate (converted from Fe2O3 particles) medium were similar to those with commercial ferric ammonium citrate medium. The biomass and lipid productivities of Chlorella PY-ZU1 cultivated with 5 mg L-1 Fe-Citrate medium were 1.30 and 1.72 times, respectively, higher than those with FeCl3 growth medium.

To determine the underlying causes of increased lipid productivity in Chlorella vulgaris, cells were grown in nitrogen-starved phosphorus-replete (N-P+) conditions, and oxidative phosphorylation pathways, luxury phosphorus uptake, photosynthetic characteristics, and lipid productivity were investigated. A maximum lipid productivity of 82.0 mg L-1 day(-1) was obtained under N-P+ conditions, which was 4.6-fold and 1.5fold higher than that obtained under nitrogen and phosphorus-replete and N-Plim conditions, respectively. Genes involved in oxidative phosphorylation, including ATP synthase (fold change = 38.74), ATP phosphohydrolase (fold change = 18.19), inorganic pyrophosphatase (fold change = 25.94), and NADH dehydrogenase, were primarily upregulated in N-P+ conditions. The ATP and total ATPase contents in cells were greater under N-P+ conditions than under control conditions, which suggests there may be a greater energy supply for lipid biosynthesis under N-P+ conditions. P-31 NMR spectra results indicate that phosphorus was luxuriously assimilated by cells under N-P+ conditions and was mainly stored as pyrophosphate, polyphosphate, orthophosphate, and monoesters. The maximum quantum efficiency and relative electron transport rate of C. vulgaris cultivated under N-P+ conditions were 0.55 and 23, respectively, which were greater than under nitrogen-starved phosphorus-limited (N-Plim) conditions. Nitrogen starvation and phosphorus repletion is undoubtedly an optimal strategy for lipid accumulation.

To promote biomass and lipid productivity of microalgae in a continuous two-stage cultivation process with 15% CO2, adequate phosphorus was supplemented in the second stage of cultivation involving nitrogen starvation and stored with regulated existence patterns to improve the metabolic activity of Chlorella PY-ZU1. The results showed that the applied strategy allowed microalgae biomass concentration and lipid productivity 1.3 and 2.2 times higher, respectively, than those without phosphorus supplementation. Chlorella PY-ZU1 cells cultivated with an adequate amount of phosphorus were analyzed by electron microscopy that revealed the distribution of polyphosphates in the cytoplasm, vacuoles, and along the cell membranes. P-31 nuclear magnetic resonance (P-31 NMR) showed polyphosphate, pyrophosphate, and orthophosphate contents of 0.59, 2.46, and 8.21 mg g(-1), respectively, in the extracts of Chlorella PY-ZU1 cells obtained by the NaOH-EDTA method. Moreover, the ATP synthase involved in the photosynthesis and oxidative phosphorylation pathways was up-regulated to promote biomass productivity, while the enzymes involved in carbon fixation and glycerolipid metabolism were up-regulated to facilitate lipid accumulation in cells.

Spermidine enhanced resistance of Chlorella to high levels of CO2 and light intensity.

For the comprehensive understanding of regulated carbon metabolism associated with lipid accumulation in microalgae Chlorella sp. cultivated with nitrogen starvation and phosphorus repletion (N-P+), the discrepancy among gene expression levels in carbon flow from CO2 to fatty acid synthesis were characterized through de novo sequencing, transcriptome assembly, annotation, and differential expression analysis. The biomass production of 1900 mg L−1 and lipid content of 677.55 mg (1010 cells)−1 in Chlorella sp. under N-P+ condition were 1.46-fold and 1.49-fold of those under nitrogen starvation and phosphorus limitation (N-Plim) condition, respectively. The expression levels of many enzymes related to carbon metabolism under N-P+ condition were upregulated, including carbon fixation, pyruvate metabolism, glycolysis, TCA cycle and fatty acid synthesis pathways. CO2 fixation accelerated through the upregulated Calvin cycle and glycolysis pathways. The rapid synthesis of pyruvate and upregulation of key rate-limiting enzymes involved in fatty acid synthesis favored lipid accumulation.

A novel tangential spiral-flow column photo-bioreactor (TSCP) was developed to strengthen the velocity field and turbulent kinetics for improving CO2 fixation with microalgae. Microalgal solution was injected into a column photobioreactor from four symmetrically arranged nozzles, whose central axes had 10 degrees-included angles with the radius. The injection solution streamline formed a tangential swirling circle, thereby driving the surrounding solution to spirally flow upward. This flow pattern was determined via computational fluid dynamics simulation and a miniature Doppler velocimeter. The optimal structural and operational parameters were determined as follows: relative diameter of imaginary tangential circle = 0.3, nozzles number = 4, and injection solution velocity = 1.33 m/s. The average turbulent kinetic energy, bubble diameter, and actual photochemical efficiency in TSCP were 15% higher, 47% lower, and 19% higher than those in the bubble column photobioreactor (BCP), respectively. Therefore, the microalgal biomass yield and maximum CO2 fixation rate in TSCP were promoted by 1.4 times and 53%, respectively.

In order to study the role of sufficient phosphorus (P) in biodiesel production by microalgae, Phaeodactylum tricornutum were cultivated in six different media treatments with combination of nitrogen (N) sufficiency/deprivation and phosphorus sufficiency/limitation/deprivation. Profiles of N and P, biomass, and fatty acids (FAs) content and compositions were measured during a 7-day cultivation period. The results showed that the FA content in microalgae biomass was promoted by P deprivation. However, statistical analysis showed that FA productivity had no significant difference (p = 0.63, >0.05) under the treatments of N deprivation with P sufficiency (N-P) and N deprivation with P deprivation (N-P-), indicating P sufficiency in N deprivation medium has little effect on increasing biodiesel productivity from P. triornutum. It was also found that the P absorption in N-P medium was 1.41 times higher than that in N sufficiency and P sufficiency (NP) medium. N deprivation with P limitation (N-P-l) was the optimal treatment for producing biodiesel from P. triornutum because of both the highest FA productivity and good biodiesel quality.

In this study, Scenedesmus obliquus NIES-2280 was cultivated heterotrophically with acetate as the carbon source. The effects of nitrogen deficiency and different phosphorus supply levels on biodiesel production by S. obliquus were investigated. It was found that S. obliquus could make good use of assimilated acetate for fatty acid accumulation. Fatty acid contents of algae in nitrogen deficiency media increased to 38-48% after 6-day cultivation. Interestingly, the productivity of fatty acid methyl esters (FAMEs) under nitrogen starved conditions increased fourfold than that under nitrogen sufficient conditions. Moreover, FAME productivity could be further enhanced by a sufficient phosphorus supply rather than under P limitation or P deficiency conditions, and the highest FAME productivity was 55.9 mg L-1 d(-1). Furthermore, the conversion yields of acetate to fatty acids (COD based) in nitrogen starvation media (18-28%) were much higher than those in nitrogen sufficient media (similar to 7%). This study indicates a great potential to combine wastewater treatment with biodiesel production via S. obliquus which can significantly improve biodiesel productivity and COD utilization under nitrogen starvation coupled with sufficient phosphorus supply. (C) 2015 Elsevier Ltd. All rights reserved.

Polyphosphate (Poly-P) accumulation has been reported in Chlorella vulgaris under nitrogen deficiency conditions with sufficient P supply, and the process has been demonstrated to have great impact on lipid productivity. In this article, the utilization of polyphosphates and the regreening process under N resupplying conditions, especially for lipid production reviving, were investigated. This regreening process was completed within approximately 3–5 days. Polyphosphates were first degraded within 3 days in the regreening process, with and without an external P supply, and the degradation preceded the assimilation of phosphate in the media with an external P offering. Nitrate assimilation was markedly influenced by the starvation of P after polyphosphates were exhausted in the medium without external phosphates, and then the reviving process of biomass and lipid production was strictly impeded. It is, thus, reasonable to assume that simultaneous provision of external N and P is essential for overall biodiesel production revival during the regreening process.

In this study the heterotrophic cultivation of Chlorella vulgaris NIES-227 fed with glucose was investigated systematically using six media types; combinations of nitrogen repletion/depletion and phosphorus repletion/limitation/depletion. It was found that a high yield of fatty acids (0.88 of fed glucose-COD) and a high content of fatty acid methyl esters (FAMEs) (89% of dry weight) were obtained under nitrogen starved conditions. To our knowledge it is the first report on such high COD conversion yield and FAME content in microalgae. The dominant fatty acid (>50%) was methyl oleate (C18:1), a desirable component for biodiesel synthesis. FAME content under nitrogen starved conditions was significantly higher than under nitrogen sufficient conditions, while phosphorus had no significant influence, indicating that nitrogen starvation was the real "fatty acids trigger" in heterotrophic cultivation. These findings could simplify the downstream extraction process, such as the extrusion of oil from soybeans, and could reduce operating costs by improving the fatty acid yield from waste COD.

In order to study the effect of phosphorus on biodiesel production from Scenedesmus obliquus especially under nitrogen deficiency conditions, six types of media with combinations of nitrogen repletion/depletion and phosphorus repletion/limitation/depletion were investigated in this study. It was found that nitrogen starvation compared to nitrogen repletion enhanced biodiesel productivity. Moreover, biodiesel productivity was further strengthened by varying the supply level of phosphorus from depletion, limitation, through to repletion. The maximum FAMEs productivity of 24.2 mg/L/day was obtained in nitrogen depletion with phosphorus repletion, which was two times higher than that in nutrient complete medium. More phosphorus was accumulated in cells under the nitrogen starvation with sufficient phosphorus condition, but no polyphosphate was formed. This study indicated that nitrogen starvation plus sufficient P supply might be the real "lipid trigger". Furthermore, results of the current study suggest a potential application for utilizing microalgae to combine phosphorus removal from wastewater with biodiesel production.

Microalgal biodiesel is an alternative bioenergy for the future. Nitrogen deprivation is usually used to increase lipid content in microalgae, however, it also lowers biomass production, resulting in not much increase of lipid productivity. Our previous study found that phosphorus played an important role in enhancing biodiesel productivity of C. vulgaris FACHB-1072 under nitrogen deficient condition. The aim of this study was to optimize two significant parameters of CO2 concentration (0.03, 4, 6, 12 %) and light intensity (40, 120, 200 μmol photons m-2 s-1) with respect to biodiesel productivity and P uptake rate of C. vulgaris FACHB-1072. It was found that the optimized conditions were 4 % CO2 concentration and 200 μmol photons m-2 s-1 light intensity. The maximum biodiesel productivity was 34.56 mg L-1 day-1; 2.7 times higher than the control (nutrient sufficient condition). Phosphorus was accumulated as polyphosphate and its maximum uptake rate was 2.08 mg L-1 day-1; twice that of the control. After optimization, the performances under nitrogen deficiency were significantly better compared with those under nitrogen sufficiency, which were rarely reported in literature. Our findings suggest a great potential to combine phosphorus removal from wastewater with biodiesel production via microalgae.

To investigate the role of phosphorus in lipid production under nitrogen starvation conditions, five types of media possessing different nitrogen and phosphorus concentrations or their combination were prepared to culture Chlorella vulgaris. It was found that biomass production under nitrogen deficient condition with sufficient phosphorus supply was similar to that of the control (with sufficient nutrition), resulting in a maximum lipid productivity of 58.39mg/L/day. Meanwhile, 31P NMR showed that phosphorus in the medium was transformed and accumulated as polyphosphate in cells. The uptake rate of phosphorus in cells was 3.8 times higher than the uptake rate of the control. This study demonstrates that phosphorus plays an important role in lipid production of C. vulgaris under nitrogen deficient conditions and implies a potential to combine phosphorus removal from wastewater with biodiesel production via microalgae.