Mechanisms of copper and zinc bioremoval by microalgae and bacteria grown in nutrient rich wastewaters.

Swine farming produces large quantities of nutrient-rich wastewater, which often contains metals such as Cu and Zn, used as feed additives for pigs. These metals must be removed from the wastewater before discharge but their retention in the biomass can limit its subsequent utilization. Photobioreactors are a very promising alternative for swine wastewater treatment, as the consortium of microalgae and bacteria growing symbiotically in these reactors allows high nutrient and metal removal efficiency at moderate costs. This work studies the mechanisms of removal of Cu(II) and Zn(II) by the two types of microorganisms growing in these photobioreactors. A microalga commonly used in wastewater treatment (Scenedesmus almeriensis) and an activated sludge were kept in contact with synthetic wastewater containing 100 mg/L of Cu and Zn. After 72 h, Scenedesmus almeriensis removed 43% of Cu and 45% of Zn, while activated sludge removed 78% of Cu and 96% of Zn. Single and sequential extractions of the biomasses using different extracting reagents revealed that biosorption on protonable groups is the dominant removal mechanisms. Mild reagents solubilized 69% of Cu and 94% of Zn from the microalgae and 76% of Cu and 93% of Zn from the activated sludge. Low metal concentrations in the oxidizable and residual fractions evidenced minimal bioaccumulation inside the cells. FTIR and ESEM-EDX analysis confirmed biosorption by ion exchange and complexation as the main metal remediation mechanisms. The weak bonds of the biosorbed Cu and Zn ions are beneficial for the valorization of biomass and the obtaining of safe bioproducts.

Removal of contaminants of emerging concern from pig manure in different operation stages of a thin-layer cascade photobioreactor. Relationship with concentrations in microalgae and manure.

The performance of a pilot-scale thin-layer cascade photobioreactor, operated in semicontinuous mode, for the removal of veterinary drug residues and other contaminants of emerging concern (CECs) from pig manure has been assessed in six operation stages. Chlorella sp. (70-90%), Scenedesmus sp. (10-25%) and Diatomea (<5%) comprise the microalgae species present during the stages. The global performance to remove the total CEC content in the photobioreactor effluent varied from 62 to 86% on each stage, while an CEC mean amount close to 8% was accumulated in the photobioreactor biomass. A relation with weather conditions was not observed. Elimination ratio was not related to the concentration in the influent which reached up to 8000 ng L-1 for some CECs. As expected, the concentrations of veterinary drugs were higher than those of non-veterinary CECs. The concentrations accumulated in the grown biomass were relative low, lower than 10 ng per fresh g excepting for a few cases. However, statistical data suggested that the linkage of CECs to microalgae biomass boosted their removal from the influent. Furthermore, it was observed that the manure liquid phase contained higher amounts of CECs than the solid phase.

Agricultural products from algal biomass grown in piggery wastewater: A techno-economic analysis.

The intensification of livestock activities lead to an increase in waste generation with high content of nutrients, as is the case of piggery wastewater. However, this type of residue can be used as culture media for algae cultivation in thin-layer cascade photobioreactors to reduce its environment impact and produce a valorizable algal biomass. Biostimulants were produced by enzymatic hydrolysis and ultrasonication of microalgal biomass, using membranes (Scenario 1) or centrifugation (Scenario 2) as harvesting methods. The co-production of biopesticides by solvent extraction was also evaluated using membranes (Scenario 3) or centrifugation (Scenario 4). The four scenarios were analyzed by a technoeconomic assessment estimating the total annualized equivalent cost and the production cost, i.e., the minimum selling price. Centrifugation provided biostimulants approximately 4 times more concentrated than membranes, but with higher expense due to the cost of the centrifuge (contribution of 62.2 % in scenario 2) and the electricity requirements. The biopesticide production resulted the highest contribution to investment cost in scenarios 3 and 4 (34 % and 43 % respectively). The use of membranes was also more advantageous to produce biopesticides, although it was 5 times more diluted than using centrifuge. The biostimulant production cost was 65.5 €/m3 with membranes and 342.6 €/m3 by centrifugation and the biopesticide production cost was 353.7 €/m3 in scenario 3 and 2,122.1 €/m3 in scenario 4. Comparing the treatment of 1 ha of land, the cost of the biostimulant produced in the four scenarios was lower than the commercial one (48.1 %, 22.1 %, 45.1 % and 24.2 % respectively). Finally, using membranes for biomass harvesting allowed economically viable plants with lower capacity and longer distance for biostimulant distribution (up to 300 km) than centrifuge (188 km). The algal biomass valorization for agricultural products production is an environmentally and economically feasible process with the adequate capacity of the plant and distribution distance.

Determination of contaminants of emerging concern in raw pig manure as a whole: difference with the analysis of solid and liquid phases separately.

The content of veterinary drugs in manure is usually estimated by the amount of residues determined in its solid or liquid phase, individually, which previously required a separation step. As an alternative, a multiresidue method for the analysis of 48 veterinary drugs and other contaminants of emerging concern (CECs) in swine raw manure as a whole has been developed and in-house validated in this work. The impact of several experimental factors during ultrasound assisted extraction was assessed. Hence, the use of alumina seemed to especially decrease the matrix effect and improve the overall recovery of drugs, mainly those with a high octanol-water partition coefficient. CECs in the extracts were analyzed by ultra-high performance liquid chromatography coupled to mass spectrometry in tandem. A standard addition-matrix matched calibration was used for quantification. Application of the method to two related samples (raw manure and farm centrifuged raw manure) from a facility revealed that the concentrations of CECs determined in the raw manure by the comprehensive methodology were higher than those calculated by adding the concentrations measured in the solid and liquid phases, separately. This was attributed to the loss of CECs adsorbed on fine particles in the suspension during the sample preparation procedure of the liquid-phase. Furthermore, the decrease of residues in the raw manure when this is centrifuged in the farm to yield compost is shown.

Determination of veterinary drugs in microalgae biomass from photobioreactors fed with piggery wastewater.

Concentration data of veterinary drugs in microalgae biomass collected from photobioreactors fed with piggery wastewaters are presented for the first time in this work. To this aim, a QuEChERS methodology and an ultrasound-assisted solid-liquid extraction have been assessed as sample preparation procedures with the purpose of determining 20 veterinary drugs, mainly antibiotics of different physico-chemical properties in addition to dexamethasone, fenbendazole and progesterone. Some critical operation parameters of the QuEChERS procedure were optimized by an experimental design but tetracycline, oxytetracycline, doxycycline, marbofloxacin and ciprofloxacin were not detected by the QuEChERS sample preparation. The use of a longer and thorough approach, a solid-liquid extraction with water/methanol in presence of primary secondary amine as a clean-up agent followed by solid-phase extraction on Oasis HLB cartridges, is recommended to monitor all intended analytes. The determination in extracts is carried out by ultra-high performance liquid chromatography-tandem mass spectrometry in selected reaction monitoring mode. Limits of detection about 0.2-42 ng per g of lyophilized microalgae sample, and repeatabilities about 6-46% (n = 5, RSDs) are reached. The solid-liquid extraction method was applied to microalgae biomass samples collected from a photobioreactor. Nine drugs were detected in the samples at relatively low concentration and a proportional relationship between the found concentrations and the octanol/water partition coefficients of the drugs has been outlined. Moreover, a linear ratio between the concentrations measured in biomass and effluent has been observed for most of the drugs.

Effect of process parameters on the valorization of components from microalgal and microalgal-bacteria biomass by enzymatic hydrolysis.

Photobioreactors for wastewater treatment coupled with nutrient recovery from the biomass is a promising biorefinery platform but requires working with microalgae-bacteria consortia. This work compares the effect that hydrolysis time and different enzymes have on the solubilization and recovery of components from microalgae-bacteria grown in piggery wastewater and microalgae grown in synthetic media by enzymatic hydrolysis. Higher carbohydrate solubilizations were obtained from microalgae-bacteria than from pure microalgae (38.5% vs. 27% Celluclast, 5 h), as expected from the SEM images. Proteases solubilized xylose remarkably well, but xylose recovery was negligible in all experiments. Alcalase hydrolysis (5 h) provided the highest peptide recovery from both biomasses (≈34%), but the peptide sizes were lower than 10 kDa. Low peptide recoveries (<20%) but larger peptide sizes (up to 135 kDa) were obtained with Protamex. Pure microalgae resulted in remarkably higher losses, but similar amino acid profiles and peptide sizes were obtained from both biomasses.

Determination of antibiotics and other veterinary drugs in the solid phase of pig manure.

The presence of residues of veterinary pharmaceuticals in farm wastewaters such as pig slurry represents a problem that needs to be tackled to avoid further contamination of environmental waters and the development of resistant bacteria. For their monitoring and control, it is necessary the existence of reliable analytical tools. The present paper describes for the first time the development and optimization by statistical experimental design of a specifically designed analytical method for the analysis of 21 veterinary drugs, including 18 antibiotics of several families (ß-lactams, tetracyclines, fluoroquinolones, sulfonamides, macrolides, among others), 1 antiparasitic, 1 analgesic and 1 hormone, in a complex environmental matrix such as the fresh solid phase of pig slurry. The resulting method, consisted of an ultrasound assisted extraction (UAE) combined with in-situ dispersive solid phase extraction (d-SPE) from a 0.3 g of freeze-dried sample aliquot followed by a preconcentration step by compact solid phase extraction (c-SPE) and subsequent instrumental analysis by ultra-high-performance-liquid-chromatography (UHPLC) coupled to mass spectrometry in tandem (MS/MS) by a triple quadrupole, was successfully validated as a very sensitive (method limit of quantification in the low ng g-1) and reliable method (relative recoveries around 100% and method repeatability featured by a general relative standard deviation below 20%). Provided raw data was intended to be processed by matrix-matched quantification approach. The resulting methodology was applied to the characterization of several pig manures from different Spanish farms sampled across breeding season between 2018 and 2019. Sample precedence showed to have a high impact in the positives, its frequency and concentration.

Influence of organic matter and CO2 supply on bioremediation of heavy metals by Chlorella vulgaris and Scenedesmus almeriensis in a multimetallic matrix.

This research evaluated the influence of organic matter (OM) and CO2 addition on the bioremediation potential of two microalgae typically used for wastewater treatment: Chlorella vulgaris (CV) and Scenedesmus almeriensis (SA). The heavy metal (HM) removal efficiencies and biosorption capacities of both microalgae were determined in multimetallic solutions (As, B, Cu, Mn, and Zn) mimicking the highest pollutant conditions found in the Loa river (Northern Chile). The presence of OM decreased the total biosorption capacity, specially in As (from 2.2 to 0.0 mg/g for CV and from 2.3 to 1.7 mg/g for SA) and Cu (from 3.2 to 2.3 mg/g for CV and from 2.1 to 1.6 mg/g for SA), but its influence declined over time. CO2 addition decreased the total HM biosorption capacity for both microalgae species and inhibited CV growth. Finally, metal recovery using different eluents (HCl, NaOH, and CaCl2) was evaluated at two different concentrations. HCl 0.1 M provided the highest recovery efficiencies, which supported values over 85% of As, 92% of Cu, and ≈100% of Mn and Zn from SA. The presence of OM during the loaded stage resulted in a complete recovery of As, Cu, Mn, and Zn when using HCl 0.1 M as eluent.

Photobioreactors based on microalgae-bacteria and purple phototrophic bacteria consortia: A promising technology to reduce the load of veterinary drugs from piggery wastewater.

Traditional swine manure treatments are not fully effective in the removal of veterinary drugs. Moreover, they are costly and entail a significant carbon footprint in many cases. Innovative biological approaches based on phototrophic microorganisms have recently emerged as promising alternatives to overcome those limitations. This work evaluated the removal of 19 veterinary drugs (i.e., 16 antibiotics, 1 analgesic, 1 anti-parasitic and 1 hormone) from piggery wastewater (PWW) in two open photobioreactors (PBR) operated with a consortium of microalgae-bacteria (AB-PBR) and purple photosynthetic bacteria (PPB-PBR). Multiple hydraulic retention times (HRT), in particular 11, 8 and 4 days, were tested during stage I, II and III, respectively. Ten out of 19 target compounds were detected with inlet drug concentrations ranging from 'non-detected' (n.d.) to almost 23,000 ng L-1 for the antibiotic oxytetracycline. Moreover, three of the antibiotics (i.e., enrofloxacin, sulfadiazine and oxytetracycline) were found at concentrations above the analytical linearity range in some or all of the samples under study. AB-PBR supported higher removal efficiencies (REs) than PPB-PBR, except for danofloxacin. Overall, REs progressively decreased when decreasing the HRT. The highest REs (>90%) were observed for doxycycline (95 ±â€¯3%) and oxytetracycline (93 ±â€¯3%) in AB-PBR during stage I. The other drugs, except sulfadimidine that was the most recalcitrant, showed REs above 70% during stage I in the same photobioreactor. In contrast, no removal was observed for danofloxacin in AB-PBR during stage III, sulfadimidine in PPB-PBR during stage III or marbofloxacin in PPB-PBR during the entire experiment.

A systematic comparison of the potential of microalgae-bacteria and purple phototrophic bacteria consortia for the treatment of piggery wastewater.

This study evaluated the performance of two open-photobioreactors operated with microalgae-bacteria (PBR-AB) and purple photosynthetic bacteria (PBR-PPB) consortia during the treatment of diluted (5%) piggery wastewater (PWW) at multiple hydraulic retention times (HRT). At a HRT of 10.6 days, PBR-AB provided the highest removal efficiencies of nitrogen, phosphorus and zinc (87 ±â€¯2, 91 ±â€¯3 and 98 ±â€¯1%), while the highest organic carbon removals were achieved in PBR-PPB (87 ±â€¯4%). The decrease in HRT from 10.6, to 7.6 and 4.1 day caused a gradual deterioration in organic carbon and nitrogen removal, but did not influence the removal of phosphorus and Zn in both photobioreactors. The decrease in HRT caused a severe wash-out of microalgae in PBR-AB and played a key role in the structure of bacterial population in both photobioreactors. In addition, batch biodegradation tests at multiple PWW dilutions (5, 10 and 15%) confirmed the slightly better performance of algal-bacterial systems regardless of PWW dilution.

Comparative uptake study of arsenic, boron, copper, manganese and zinc from water by different green microalgae.

This work represents a comparative uptake study of the toxic elements arsenic, boron, copper, manganese and zinc in monometallic and multimetallic solutions by four green microalgae species (Chlamydomonas reinhardtii, Chlorella vulgaris, Scenedesmus almeriensis and an indigenous Chlorophyceae spp.), evaluating the effect of pH and contact time. Maximum removal efficiencies for each toxic element were 99.4% for Mn (C. vulgaris, pH 7.0, 3 h), 91.9% for Zn (Chlorophyceae spp., pH 5.5, 3 h), 88% for Cu (Chlorophyceae spp., pH 7.0, 10 min), 40.7% for As (S. almeriensis, pH 9.5, 3 h) and 38.6% for B (S. almeriensis, pH 5.5, 10 min). B removal efficiencies decreased remarkably in multimetallic solutions (down to 0.2% in C. reinhardtii), except for Chlorophyceae spp., the only species isolated from a polluted environment. FTIR spectra shown the highest interactions for As (1150-1300 cm-1) and Cu (3300, 1741, 1535, 1350-1400 cm-1). Results confirm microalgae biomass as a potential biosorbent for toxic elements.

Evaluation of the dynamics of microalgae population structure and process performance during piggery wastewater treatment in algal-bacterial photobioreactors.

The dynamics of microalgae population during piggery wastewater (PWW) treatment in four open photobioreactors operated at 27days of hydraulic retention time, and inoculated with Chlorella sp. (R1), Acutodesmus obliquus (R2), Oscillatoria sp. (R3) and in the absence of inoculum (R4), were evaluated for 6months. In addition, the algal-bacterial biomass concentration, removal of organic matter, nutrients and heavy metals were also assessed. The results revealed a high diversity and rapid variations in the structure of microalgae populations, Chlorella sp. being dominant in R4 throughout most of the operational period. Steady state average biomass concentration ranged from 2445-2610mg/L in R1-R3 to 3265mg/L in R4. No significant differences were recorded in the removal efficiencies (REs) of total organic carbon (86-87%), inorganic carbon (62-71%), total nitrogen (82-85%) and total phosphorous (90-92%). Finally, Zn-REs accounted for 26% in R3, 37% in R2, and 49% in R1 and R4.

Comparative evaluation of piggery wastewater treatment in algal-bacterial photobioreactors under indoor and outdoor conditions.

This work evaluated the performance of four open algal-bacterial photobioreactors operated at ≈26days of hydraulic retention time during the treatment of 10 (×10) and 20 (×20) times diluted piggery wastewater (PWW) under indoor (I) and outdoor (O) conditions for four months. The removal efficiencies (REs) of organic matter, nutrients and zinc from PWW, along with the dynamics of biomass concentration and structure of algal-bacterial population were assessed. The highest TOC-RE, TP-RE and Zn-RE (94±1%, 100% and 83±2%, respectively) were achieved indoors in ×10 PWW, while the highest TN-RE (72±8%) was recorded outdoors in ×10 PWW. Chlorella vulgaris was the dominant species regardless of the ambient conditions and PWW dilution. Finally, DGGE-sequencing of the bacterial community revealed the occurrence of four phyla, Proteobacteria being the dominant phylum with 15 out of the 23 most intense bands.

Effect of four pretreatments on enzymatic hydrolysis and ethanol fermentation of wheat straw. Influence of inhibitors and washing.

Pretreatment is essential in the production of alcohol from lignocellulosic material. In order to increase enzymatic sugar release and bioethanol production, thermal, dilute acid, dilute basic and alkaline peroxide pretreatments were applied to wheat straw. Compositional changes in pretreated solid fractions and sugars and possible inhibitory compounds released in liquid fractions were analysed. SEM analysis showed structural changes after pretreatments. Enzymatic hydrolysis and fermentation by Pichia stipitis of unwashed and washed samples from each pretreatment were performed so as to compare sugar and ethanol yields. The effect of the main inhibitors found in hydrolysates (formic acid, acetic acid, 5-hydroxymethylfurfural and furfural) was first studied through ethanol fermentations of model media and then compared to real hydrolysates. Hydrolysates of washed alkaline peroxide pretreated biomass provided the highest sugar concentrations, 31.82g/L glucose, and 13.75g/L xylose, their fermentation yielding promising results, with ethanol concentrations reaching 17.37g/L.

Sugarcane bagasse ozonolysis pretreatment: effect on enzymatic digestibility and inhibitory compound formation.

Sugarcane bagasse was pretreated with ozone to increase lignocellulosic material digestibility. Bagasse was ozonated in a fixed bed reactor at room temperature, and the effect of the two major parameters, ozone concentration and sample moisture, was studied. Acid insoluble and total lignin decreased whereas acid soluble lignin increased in all experiments. Pretreatment barely attacked carbohydrates, with cellulose and xylan recovery rates being >92%. Ozonolysis increased fermentable carbohydrate release considerably during enzymatic hydrolysis. Glucose and xylose yields increased from 6.64% and 2.05%, for raw bagasse, to 41.79% and 52.44% under the best experimental conditions. Only xylitol, lactic, formic and acetic acid degradation compounds were found, with neither furfural nor HMF (5-hydroxymethylfurfural) being detected. Washing detoxification provided inhibitor removal percentages above 85%, increasing glucose hydrolysis, but decreasing xylose yield by xylan solubilization. SEM analysis showed structural changes after ozonization and washing.

An analysis of lignin removal in a fixed bed reactor by reaction of cereal straws with ozone.

The chemical pretreatment with ozone of rye and wheat straws was carried out in a fixed bed reactor. The effect of ozone pretreatment time on lignin removal was determined. Glucose and xylose concentrations in the hydrolysates were also measured after the subsequent enzymatic hydrolysis step. Acid insoluble lignin reacts with ozone within the first 90 min reaction. Insoluble lignin reduction was about 50%. The higher hydrolysis yields were obtained after 120 min ozonation: the glucose yield ranged from 40% to 50% for rye straw and from 34% to 39% for wheat straw, whereas xylose yields were about 30%, independently of the cereal straw. The glucose yields corresponding to the untreated raw straws were considerably lower, about 10%. Longer ozonation time sharply reduced the production of monosaccharides, probably due to the formation of side products. The kinetic model, with the reaction parameters estimated, predicted reasonably well the experimental data.

Effect of inhibitors formed during wheat straw pretreatment on ethanol fermentation by Pichia stipitis.

The inhibitory effect of the main inhibitors (acetic acid, furfural and 5-hydroxymethylfurfural) formed during steam explosion of wheat straw was studied through ethanol fermentations of model substrates and hydrolysates from wheat straw by Pichia stipitis. Experimental results showed that an increase in acetic acid concentration led to a reduction in ethanol productivity and complete inhibition was observed at 3.5 g/L. Furfural produced a delay on sugar consumption rates with increasing concentration and HMF did not exert a significant effect. Fermentations of the whole slurry from steam exploded wheat straw were completely inhibited by a synergistic effect due to the presence of 1.5 g/L acetic acid, 0.15 g/L furfural and 0.05 g/L HMF together with solid fraction. When using only the solid fraction from steam explosion, hydrolysates presented 0.5 g/L of acetic acid, whose fermentations have submitted promising results, providing an ethanol yield of 0.45 g ethanol/g sugars and the final ethanol concentration reached was 12.2 g/L (10.9 g ethanol/100 g DM).

Effect of ozonolysis pretreatment on enzymatic digestibility of wheat and rye straw.

Wheat and rye straws were pretreated with ozone to increase the enzymatic hydrolysis extent of potentially fermentable sugars. Through a 2(5-1) factorial design, this work studies the influence of five operating parameters (moisture content, particle size, ozone concentration, type of biomass and air/ozone flow rate) on ozonization pretreatment of straw in a fixed bed reactor under room conditions. The acid insoluble lignin content of the biomass was reduced in all experiments involving hemicellulose degradation. Near negligible losses of cellulose were observed. Enzymatic hydrolysis yields of up to 88.6% and 57% were obtained compared to 29% and 16% in non-ozonated wheat and rye straw respectively. Moisture content and type of biomass showed the most significant effects on ozonolysis. Additionally, ozonolysis experiments in basic medium with sodium hydroxide evidenced a reduction in solubilization and/or degradation of lignin and reliable cellulose and hemicellulose degradation.