Microalgae-Based Biorefineries: Challenges and Future Trends to Produce Carbohydrate Enriched Biomass, High-Added Value Products and Bioactive Compounds.

Microalgae have demonstrated a large potential in biotechnology as a source of various macromolecules (proteins, carbohydrates, and lipids) and high-added value products (pigments, poly-unsaturated fatty acids, peptides, exo-polysaccharides, etc.). The production of biomass at a large scale becomes more economically feasible when it is part of a biorefinery designed within the circular economy concept. Thus, the aim of this critical review is to highlight and discuss challenges and future trends related to the multi-product microalgae-based biorefineries, including both phototrophic and mixotrophic cultures treating wastewater and the recovery of biomass as a source of valuable macromolecules and high-added and low-value products (biofertilizers and biostimulants). The therapeutic properties of some microalgae-bioactive compounds are also discussed. Novel trends such as the screening of species for antimicrobial compounds, the production of bioplastics using wastewater, the circular economy strategy, and the need for more Life Cycle Assessment studies (LCA) are suggested as some of the future research lines.

Pontederia sagittata and Cyperus papyrus contribution to carbon storage in floating treatment wetlands established in subtropical urban ponds.

Carbon sequestration is considered an ecosystem service of regulation provided by diverse ecosystems, including wetlands. It has been widely evaluated in the soil of natural wetlands while in constructed wetlands, there is scanty information. In Floating Treatment Wetlands (FTW) there is none. Previously, our research group reported the efficient performance of FTW in an urban polluted pond for two years. As a follow up, the aim of this work was to investigate the contribution of Cyperus papyrus and Pontederia sagittata to carbon storage (CS) in four FTW established in eutrophic urban ponds in a subtropical region. Plant growth, productivity, and CS were assessed in the aboveground biomass of C. papyrus and P. sagittata and the belowground biomass (root mix from C. papyrus and P. sagittata), throughout 26 months in 2 FTW with an area of 17.5 m2 (FTW1) and 33 m2 (FTW2) and throughout 19 months in 2 FTW with an area of 25 m2 (FTW3) and 33 m2 (FTW4), respectively. The macrophyte growth depended on various factors, such as the season, the plant species, and the location of the FTW. High relative growth rate values were found for both species (0.125 and 0.142 d-1 for P. sagittata and C. papyrus, respectively), especially during summer and early autumn. The highest values of productivity were 337 ± 125 gdw m-2d-1 for the aboveground biomass of C. papyrus in FTW2, 311 ± 96.90 gdwm-2d-1 for the aboveground of P. sagittata in FTW1, and 270 ± 107 gdw m-2d-1 for the belowground biomass in FTW2. The mean values of CS for P. sagittata found in FTW1 were 1.90 ± 0.94 kg m-2, while for C. papyrus in FTW2 they were 4.09 ± 0.73 kg m-2. The contribution of the belowground biomass to CS was also significant in FTW2 (4.58 ± 0.59 kg m-2).

Light Intensity and Nitrogen Concentration Impact on the Biomass and Phycoerythrin Production by Porphyridium purpureum.

Several factors have the potential to influence microalgae growth. In the present study, nitrogen concentration and light intensity were evaluated in order to obtain high biomass production and high phycoerythrin accumulation from Porphyridium purpureum. The range of nitrogen concentrations evaluated in the culture medium was 0.075-0.450 g L-1 and light intensities ranged between 30 and 100 µmol m-2 s-1. Surprisingly, low nitrogen concentration and high light intensity resulted in high biomass yield and phycoerythrin accumulation. Thus, the best biomass productivity (0.386 g L-1 d-1) and biomass yield (5.403 g L-1) were achieved with NaNO3 at 0.075 g L-1 and 100 µmol m-2 s-1. In addition, phycoerythrin production was improved to obtain a concentration of 14.66 mg L-1 (2.71 mg g-1 of phycoerythrin over dry weight). The results of the present study indicate that it is possible to significantly improve biomass and pigment production in Porphyridium purpureum by limiting nitrogen concentration and light intensity.

Continuous dye adsorption and desorption on an invasive macrophyte (Salvinia minima).

The continuous adsorption-desorption of methylene blue (MB) on an invasive macrophyte, Salvinia minima, was investigated in fixed-bed columns. The effects of bed depth (h) (9.30, 18.70, and 28 cm), inlet dye concentration (C0) (51 ± 1.20, 154 ± 2.00, and 250 ± 1.50 mg L-1), and flow rate (Q) (7 and 14 mL min-1) on dye removal and breakthrough curves were assessed. Thomas, modified dose-response (MDR) and bed depth service time (BDST) models were fitted to the experimental data. Desorption and regeneration studies were also performed. The breakthrough time was affected by h, C0, and Q. The dynamic bed capacity at the breakthrough point (qb) increased with increasing h but decreased with increasing C0 and Q. Dynamic bed capacities (qe) from 318 to 322 mg g-1 were achieved at h = 28 cm, C0 = 154 ± 2.0, or 250 ± 1.50 mg L-1, independently of the Q value. High MB removals were also observed (75-78%). FTIR analysis revealed that hydroxyl and carboxyl groups could be involved in dye adsorption. MDR and BDST models were both successfully used to predict the breakthrough curves of MB adsorption onto S. minima. A high regeneration efficiency (> 87%) was obtained after three adsorption-desorption cycles. These results confirm that the use of S. minima biomass could be a very efficient and eco-friendly alternative for MB adsorption in continuous mode.

Carbon speciation and flocculation in Neochloris oleoabundans cultures using anaerobically digested stillage.

The effects of bicarbonate loading rate (BLR) and pH on growth kinetics, inorganic carbon speciation, carbon fixation and lipid content in Neochloris oleoabundans cultures using anaerobically digested stillage (ADS) (2% v/v) were investigated. Four different cultures were established: culture A with BLR = 1 g l-1 day-1 and no pH adjustment, culture B with BLR = 0.5 g l-1 day-1 and no pH adjustment, culture C with BLR = 1 g l-1 day-1 and pH adjustment at 7.0, and culture D with BLR = 0.5 g l-1 day-1 and pH adjustment at 7.0. The experiments were carried out in flat plate reactors (4 l) at controlled conditions (light intensity of 134 µmol photon m-1 s-1 and photoperiod 16 light/8 darkness; temperature of 32 ± 1 °C). The effects of pH (7, 10.41, 10.65, and 12), time (15, 30, 60, and 90 min), and concentration of a cationic polyelectrolyte (CP) (10 and 20 mg l-1) on the flocculation efficiency (FE) of N. oleoabundans were also investigated. The results showed that bicarbonate was the predominant carbon species in the media and the main carbon source for microalgae growth in all cultures. The highest productivity (87.70 ± 9.70 mg l-1 day-1) and CO2(aq) fixation rate (0.15 g CO2(aq) l-1 day-1) were found in culture B. The lipid content in N. oleoabundans was affected negatively by the pH adjustment at 7.0 during its growth; higher values were found in cultures with no pH adjustment (37.10% and 38.85% dw for culture A and B, respectively) as compared to those obtained in cultures with pH adjustment (27.35% and 22.20% dw for culture C and D, respectively) (p < 0.05). Regarding flocculation, the addition of 20 mg CP l-1 was required to obtain a FE > 95% in cultures A and B, although a significant FE (40-59%) occurred without CP addition at a high pH (≥ 10.41) in all cultures.

Pilot-scale outdoor production of Scenedesmus sp. in raceways using flue gases and centrate from anaerobic digestion as the sole culture medium.

This work investigated the production of Scenedesmus sp. in semi-continuous mode in three pilot-scale outdoor raceways (7.2 m2) using flue gas for CO2 supply and centrate from the anaerobic digestion of urban wastewater as the sole nutrient source. Experiments were performed at different culture depths, 5, 10 and 15 cm, while evaluating two centrate concentrations (30% and 45%) at dilution rates of 0.2 and 0.3 d-1. Under optimal conditions of 30% centrate, 0.3 d-1 dilution rate and a 15 cm culture depth, a maximum biomass productivity of 22.9 g m-2 d-1 was obtained. The optical properties of the cultures were studied and the results showed a photosynthetic efficiency of up to 2.0% and a quantum yield of 0.3 g biomass E-1. Nitrogen and phosphorus removal rates of 3 g N m-2 d-1 and 0.6 g P m-2 d-1 were recorded, respectively. Lipid productivity of 2.3 g m-2 d-1 was determined possessing a suitable fatty acids profile for biofuel production.

Year-round phytofiltration lagoon assessment using Pistia stratiotes within a pilot-plant scale biorefinery.

Phytofiltration lagoons are phytoremediation technologies suitable for tropical and sub-tropical regions requiring cost-effective and echo-friendly technologies. A biorefinery of fourth generation has been implemented at pilot plant level in Xalapa, Mexico, and the phytofiltration lagoon, being the first module for provision of treated water and plant biomass for biofuel production plays a key role. The aim of this work was to evaluate the performance of such phytofiltration lagoon with a working volume of 13,000 L for the removal of nutrients from an urban river polluted with domestic wastewater and the biomass productivity of the macrophyte Pistia stratiotes, during five different experimental periods, comprising 42 days each one. The maximum absolute growth rates (AGR, gdwday-1) registered for P. stratiotes during the Aug-Oct '15 and the March-Apr '16 and Apr-May '16 period were in the range of 13.51±2.66 to 16.54±2.02gdwday-1. The average biomass productivity was 5.808gdwm-2day-1. Productivities were similar during the periods of Aug-Oct '15, Mar-Apr '16 and Apr-May '16 and significantly higher (p<0.05) than those registered in Oct-Nov '15 and Jan-Feb '16. Removal percentages of COD and nutrients varied according to the season. COD was in the range of 47.82±39.3% to 88.00±15.0%. Ammonium N was in the range of 76.78±21% to 98.79±0.9%. Nitrates were removed in the range of 16.92±64%. to 97.14±4.5%. Finally, phosphates were removed very effectively, from 73.72±18.5% to 92.89±4.3%. A hydraulic retention time of 7 days was enough for the effective treatment of the water from the polluted river. It was concluded that the phytofiltration lagoon with P. stratiotes is very feasible within the biorefinery for providing biomass year-round and for treating the polluted water very effectively.

Long-term assessment at field scale of Floating Treatment Wetlands for improvement of water quality and provision of ecosystem services in a eutrophic urban pond.

Pollution of urban water bodies requires stringent control measures and the development of low-cost and highly efficient alternative technologies. In contrast to Constructed Wetlands, Floating Treatment Wetlands (FTWs) have the advantage of not requiring large surface of land since they operate in situ. However, there is limited information about their long-term evaluation while operating at field scale. The aim of this work was to assess the performance of FTWs using a combination of Pontederia sagittata and Cyperus papyrus for the improvement of the water quality and provision of ecosystem services of a eutrophic urban pond. The FTWs were built with low-cost material easy to acquire and to ensemble. Two FTWs (17.5m2 and 33m2) located in Pond 1 within a complex of 4 urban artificial ponds were evaluated for two years. They promoted an increase in the dissolved oxygen (D.O.) within a range of 15 to 67%, a removal of fecal coliforms in the range of 9 to 86% and a nitrate removal in the range of 9 to 76%. The plant productivity reached a maximum of 363gdmm-2d-1 in the FTW1 and 536gdmm-2d-1 in the FTW2 during the period March-June 2016. The TKN and the TP content in the plant were in the range of 18.3 to 28.1 and of 0.05 to 0.196gkg-1 dry matter, respectively. In conclusion, the tested FTWs have proved to be a very beneficial low-cost technology for the improvement of water quality and provision of ecosystem services.

Dual purpose system that treats anaerobic effluents from pig waste and produce Neochloris oleoabundans as lipid rich biomass.

Dual purpose systems that treat wastewater and produce lipid rich microalgae biomass have been indicated as an option with great potential for production of biodiesel at a competitive cost. The aim of the present work was to develop a dual purpose system for the treatment of the anaerobic effluents from pig waste utilizing Neochloris oleoabundans and to evaluate its growth, lipid content and lipid profile of the harvested biomass and the removal of nutrients from the media. Cultures of N. oleoabundans were established in 4 L flat plate photobioreactors using diluted effluents from two different types of anaerobic filters, one packed with ceramic material (D1) and another one packed with volcanic gravel (D2). Maximum biomass concentration in D1 was 0.63 g L(-1) which was significantly higher than the one found in D2 (0.55 g L(-1)). Cultures were very efficient at nutrient removal: 98% for NNH4(+) and 98% for PO4(3-). Regarding total lipid content, diluted eflluents from D2 promoted a biomass containing 27.4% (dry weight) and D1 a biomass containing 22.4% (dry weight). Maximum lipid productivity was also higher in D2 compared to D1 (6.27±0.62 mg L(-1) d(-1) vs. 5.12±0.12 mg L(-1) d(-1)). Concerning the FAMEs profile in diluted effluents, the most abundant one was C18:1, followed by C18:2 and C16:0. The profile in D2 contained less C18:3 (linolenic acid) than the one in D1 (4.37% vs. 5.55%). In conclusion, this is the first report demonstrating that cultures of N. oleoabundans treating anaerobic effluents from pig waste are very efficient at nutrient removal and a biomass rich in lipids can be recovered. The maximum total lipid content and the most convenient FAMEs profile were obtained using effluents from a digester packed with volcanic gravel.

Cascading impacts of anthropogenically driven habitat loss: deforestation, flooding, and possible lead poisoning in howler monkeys (Alouatta pigra).

To construct informed conservation plans, researchers must go beyond understanding readily apparent threats such as habitat loss and bush-meat hunting. They must predict subtle and cascading effects of anthropogenic environmental modifications. This study considered a potential cascading effect of deforestation on the howler monkeys (Alouatta pigra) of Balancán, Mexico. Deforestation intensifies flooding. Thus, we predicted that increased flooding of the Usumacinta River, which creates large bodies of water that slowly evaporate, would produce increased lead content in the soils and plants, resulting in lead exposure in the howler monkeys. The average lead levels were 18.18 ± 6.76 ppm in the soils and 5.85 ± 4.37 ppm in the plants. However, the average lead content of the hair of 13 captured howler monkeys was 24.12 ± 5.84 ppm. The lead levels in the animals were correlated with 2 of 15 blood traits (lactate dehydrogenase and total bilirubin) previously documented to be associated with exposure to lead. Our research illustrates the urgent need to set reference values indicating when adverse impacts of high environmental lead levels occur, whether anthropogenic or natural, and the need to evaluate possible cascading effects of deforestation on primates.

Microbial fixation of CO2 in water bodies and in drylands to combat climate change, soil loss and desertification.

The growing concern for the increase of the global warming effects due to anthropogenic activities raises the challenge of finding novel technological approaches to stabilize CO2 emissions in the atmosphere and counteract impinging interconnected issues such as desertification and loss of biodiversity. Biological-CO2 mitigation, triggered through biological fixation, is considered a promising and eco-sustainable method, mostly owing to its downstream benefits that can be exploited. Microorganisms such as cyanobacteria, green algae and some autotrophic bacteria could potentially fix CO2 more efficiently than higher plants, due to their faster growth. Some examples of the potential of biological-CO2 mitigation are reported and discussed in this paper. In arid and semiarid environments, soil carbon sequestration (CO2 fixation) by cyanobacteria and biological soil crusts is considered an eco-friendly and natural process to increase soil C content and a viable pathway to soil restoration after one disturbance event. Another way for biological-CO2 mitigation intensively studied in the last few years is related to the possibility to perform carbon dioxide sequestration using microalgae, obtaining at the same time bioproducts of industrial interest. Another possibility under study is the exploitation of specific chemotrophic bacteria, such as Ralstonia eutropha (or picketii) and related organisms, for CO2 fixation coupled with the production chemicals such as polyhydroxyalkanoates (PHAs). In spite of the potential of these processes, multiple factors still have to be optimized for maximum rate of CO2 fixation by these microorganisms. The optimization of culture conditions, including the optimal concentration of CO2 in the provided gas, the use of metabolic engineering and of dual purpose systems for the treatment of wastewater and production of biofuels and high value products within a biorefinery concept, the design of photobioreactors in the case of phototrophs are some of the issues that, among others, have to be addressed and tested for cost-effective CO2 sequestration.

Heavy metals in the habitat and throughout the food chain of the Neotropical otter, Lontra longicaudis, in protected Mexican wetlands.

Top predators like the Neotropical otter, Lontra longicaudis annectens, are usually considered good bioindicators of habitat quality. In this study, we evaluated heavy metal contamination (Hg(tot), Pb, Cd) in the riverine habitat, prey (crustaceans and fish), and otter feces in two Ramsar wetlands with contrasting upstream contamination discharges: Río Blanco and Río Caño Grande in Veracruz, Mexico, during the dry, the wet, and the nortes seasons. Most comparisons revealed no differences between sites while seasonal differences were repeatedly detected for all of the compartments. Higher concentrations of Pb during the dry season and of Cd during the wet season in otter feces mirrored differences detected in the most seasonally consumed prey. Compared with fecal methylmercury values reported for the European otter (0.25-0.75 mg kg(-1)) in unprotected areas, the Hg(tot) levels that we measured were lower (0.02-0.17 mg kg(-1)). However, Pb (117.87 mg kg(-1)) and Cd (9.14 mg kg(-1)) concentrations were higher (Pb, 38.15 mg kg(-1) and Cd, 4.72 mg kg(-1)) in the two Ramsar wetlands. Protected areas may shelter species, but those with water-linked diets may suffer the effect of chemicals used upstream.

Heavy metal removal in phytofiltration and phycoremediation: the need to differentiate between bioadsorption and bioaccumulation.

Phytoremediation and phycoremediation are cost-effective and environmentally sound technologies for the treatment of polluted streams and wastewaters contaminated with metals. Currently, the most commonly used parameter to assess the metal uptake of biomass is (q) expressed as mg metal g dry weight(-1). By contrast, the bioconcentration factor (BCF) is one of the most widely used factors to evaluate the metal uptake capacity of macrophytes. However, both parameters the metal uptake (q) and the BCF cannot be applied to differentiate between the ability of live plants or photosynthetic microorganisms to adsorb the metal onto their surface through passive mechanisms or to accumulate the contaminant at intracellular level through metabolically active mechanisms. This mini review has the objective of discussing the need to differentiate between bioadsorption and bioaccumulation of metals in live plants and photosynthetic microorganisms used in phytofiltration and phycoremediation processes, respectively. The use of two specific factors, the bioadsorption factor (BAF) and the intracellular accumulation factor (IAF) that have been previously reported in order to make a clear differentiation between these two metal removal mechanisms in Salvinia minima and Leptolyngbya crossbyana is highlighted. It is suggested that the BAF and the IAF can be used in phytofiltration wetlands and phycoremediation lagoons, where there is the need of specific information indicating the fate of the metal in order to gain information about possible removal mechanisms. These factors could also provide a tool to decide whether it is possible to harvest the biomass and to recover a fair amount of metal adsorbed onto the surface by means of desorbent agents. A critical assessment of the use of EDTA as desorbent agent is also included.

Dual purpose microalgae-bacteria-based systems that treat wastewater and produce biodiesel and chemical products within a biorefinery.

Excess greenhouse gas emissions and the concomitant effect on global warming have become significant environmental, social and economic threats. In this context, the development of renewable, carbon-neutral and economically feasible biofuels is a driving force for innovation worldwide. A lot of effort has been put into developing biodiesel from microalgae. However, there are still a number of technological, market and policy barriers that are serious obstacles to the economic feasibility and competitiveness of such biofuels. Conversely, there are also a number of business opportunities if the production of such alternative biofuel becomes part of a larger integrated system following the Biorefinery strategy. In this case, other biofuels and chemical products of high added value are produced, contributing to an overall enhancement of the economic viability of the whole integrated system. Additionally, dual purpose microalgae-bacteria-based systems for treating wastewater and production of biofuels and chemical products significantly contribute to a substantial saving in the overall cost of microalgae biomass production. These types of systems could help to improve the competitiveness of biodiesel production from microalgae, according to some recent Life Cycle Analysis studies. Furthermore, they do not compete for fresh water resources for agricultural purposes and add value to treating the wastewater itself. This work reviews the most recent and relevant information about these types of dual purpose systems. Several aspects related to the treatment of municipal and animal wastewater with simultaneous recovery of microalgae with potential for biodiesel production are discussed. The use of pre-treated waste or anaerobic effluents from digested waste as nutrient additives for weak wastewater is reviewed. Isolation and screening of microalgae/cyanobacteria or their consortia from various wastewater streams, and studies related to population dynamics in mixed cultures, are highlighted as very relevant fields of research. The species selection may depend on various factors, such as the biomass and lipid productivity of each strain, the characteristics of the wastewater, the original habitat of the strain and the climatic conditions in the treatment plant, among others. Some alternative technologies aimed at harvesting biomass at a low cost, such as cell immobilization, biofilm formation, flocculation and bio-flocculation, are also reviewed. Finally, a Biorefinery design is presented that integrates the treatment of municipal wastewater with the recovery of oleaginous microalgae, together with the use of seawater supplemented with anaerobically digested piggery waste for cultivating Arthrospira (Spirulina) and producing biogas, biodiesel, hydrogen and other high added value products. Such strategies offer new opportunities for the cost-effective and competitive production of biofuels along with valuable non-fuel products.

Acetate enhances the specific consumption rate of toluene under denitrifying conditions.

Toluene is usually present in the environment as a contaminant along with other carbon sources which may influence its removal. In this work we studied the effect of a readily consumable carbon source such as acetate on toluene mineralization under denitrifying conditions. Continuous and batch cultures with stabilized denitrifying sludge were carried out. An upflow anaerobic sludge blanket reactor (UASB) was fed with several ratios of acetate-C/toluene-C loading rates (mg C/L-day: 100/0, 75/25, 50/50, and 0/100). Batch assays with different acetate-C/toluene-C ratios (10/70, 30/50, 50/30, and 65/20 mg C/L) were also done. As the acetate loading rate decreased in the culture, the carbon and nitrate consumption efficiency decreased by 40% and 34%, respectively. HCO(3) (-) and N(2) yields also decreased by 43%. Analysis of the denitrifying community using the denaturing gradient gel electrophoresis technique indicated that there was no clear relationship between its population profile and the metabolic pattern. In batch assays, when the acetate concentration was higher than that of toluene (65 mg acetate-C/L vs 20 mg toluene-C/L), the specific consumption rate of toluene (q(T)) was two times higher than in assays with 20 mg toluene-C/L as the sole electron source (0.006 mg C/mg volatile suspended solids-day). It is proposed that acetate can act by enhancing the growth of microbial populations and as a biochemical enhancer. The results show that acetate addition can be useful to improve the consumption rate of toluene in contaminated water.

Constructed wetland mesocosms for the treatment of diluted sugarcane molasses stillage from ethanol production using Pontederia sagittata.

Sugarcane molasses stillage contains a very high concentration of organic matter and toxic/recalcitrant compounds. Its improper disposal has become a global problem and there is very scanty information about its treatment using phytotechnologies. This work aimed at evaluating the performance of subsurface flow constructed wetlands (SSF CWs) mesocosms planted with Pontederia sagittata and operating at two hydraulic retention times (HRTs), compared to an unplanted SSF CWs, for the treatment of diluted stillage subjected to no pre-treatment apart from an adjustment to pH 6.0. CWs were fed with very high surface COD loading rates (i.e. 47.26 and 94.83gCOD/m(2)d). The planted CWs were able to remove COD in the range of 80.24-80.62%, BOD(5) in the range of 82.20-87.31%, TKN in the range of 73.42-76.07%, nitrates from 56-58.74% and sulfates from 68.58-69.45%, depending on the HRT. Phosphate and potassium were not removed. It was concluded that this type of CWs is a feasible option for the treatment of diluted stillage.

Leaching of lead by ammonium salts and EDTA from Salvinia minima biomass produced during aquatic phytoremediation.

Plant biomass harvested after heavy-metal phytoremediation must be considered as a hazardous waste that should be contained or treated appropriately before disposal or reuse. As a potential method to detoxify the biomass and to convert this material to a suitable fertilizer or mulch, leaching of lead (Pb) from Salvinia minima biomass was studied by testing water, several aqueous ammonium salts, and EDTA solution as lead extractants. The research was carried out in two phases: (i) a leaching study to determine the lead-extraction efficiency of the different leachants, and (ii) a thermodynamic analysis to identify the likely reactions and stable Pb(II) species formed in the leaching systems of the most efficient leachants. Experimentally, lead concentrations measured in leached biomass and in leachates were significantly different among the various leachants. It was determined that the extraction strength of the leachants followed the order: EDTA>ammonium oxalate>water approximately ammonium nitrate>ammonium acetate, achieving Pb extraction efficiencies of 99%, 70%, 7.2%, 6.9% and 1.3%, respectively, in single-stage extractions. The thermodynamic study indicated that the dominant species produced by the leaching process should be the soluble species PbEDTA2- for EDTA system, and the insoluble Pb(COO)2S precipitate for the oxalate system.