Current Concentrations of Zn, Cu, and As in Piggery Wastewater Compromise Nutrient Removals in Microalgae-Bacteria Photobioreactors Due to Altered Microbial Communities.

The treatment of pig manure is a major environmental issue, and photobioreactors containing consortia of microalgae and bacteria have proven to be a promising and sustainable treatment alternative. This work studies the effect of Cu, Zn and As, three toxic elements frequently present in piggery wastewater, on the performance and microbiome of photobioreactors. After dopage with Zn (100 mg/L), Cu (100 mg/L), and As (500 µg/L), the high biomass uptake of Zn (69-81%) and Cu (81-83%) decreased the carbon removal in the photobioreactors, inhibited the growth of Chlorella sp., and affected heterotrophic bacterial populations. The biomass As uptake result was low (19%) and actually promoted microalgae growth. The presence of Cu and As decreased nitrogen removal, reducing the abundance of denitrifying bacterial populations. The results showed that metal(loid)s significantly affected 24 bacterial genera and that they did not recover after exposure. Therefore, this study makes an important contribution on the impact of the presence of metal(loid)s in piggery wastewater that compromises the overall performance of PBRs, and so, the environmental and health impact of treated effluents.

Effect of operational parameters, environmental conditions, and biotic interactions on bacterial communities present in urban wastewater treatment photobioreactors.

The effects of water depth, operational and environmental conditions on bacterial communities were analyzed in microalgal-bacterial outdoor photobioreactors treating urban wastewaters from March to August 2014. Three raceway photobioreactors inoculated with Scenedesmus sp. and with different water depths (20, 12, and 5 cm) were used at different dilution rates (0.15, 0.3, 0.4, and 0.5 d-1). A thin-layer reactor with 2 cm water depth and operated at 0.3 d-1 was used as a control. The results showed that biomass productivity increased as water depth decreased. The highest biomass productivity was 0.196 gL-1d-1, 0.245 gL-1d-1, and 0.457 gL-1d-1 for 20, 12, and 5 cm depth raceway photobioreactors, respectively. These values were lower than the maximum productivity registered in the control reactor (1.59 gL-1d-1). Bacterial communities, analyzed by high-throughput 16S rRNA sequencing, were not affected by water depth. A decrease in community evenness was related to a decrease in nutrient removal. Hetetrotrophs and phototrophs, mainly from the family Rhodobacteraceae, dominated bacterial diversity. The community changed due to increasing temperatures, irradiance, and organic carbon, ammonia, and phosphate contents in the photobioreactor-influent as well as, microalgae inhibition and higher organic carbon in the effluent. The photobioreactors shared a core-biome that contained five clusters of co-occurring microorganisms. The bacteria from the different clusters were taxonomically and ecologically different but functionally redundant. Overall, the drivers of the community changes could be related to abiotic variables and complex biological interactions, likely mediated by microalgae excretion of organic substances and the microorganisms' competence for substrates.

Evaluation of pretreatments for solubilisation of components and recovery of fermentable monosaccharides from microalgae biomass grown in piggery wastewater.

Microalgae-bacteria biomass cultured in wastewater is an interesting renewable material capable of metabolising nutrients from wastes into carbohydrates, proteins, and lipids through photosynthesis. Despite the interest in the valorisation of this biomass to improve the viability of microalgae-based wastewater treatment processes, very scarce research has been devoted to the fractional recovery of its components. This work evaluates the effect of different pretreatments coupled with enzymatic hydrolysis on the solubilisation of biomass components and on the recovery of fermentable monosaccharides (glucose and xylose) from Scenedesmaceae based biomass grown in a thin layer reactor feed with piggery wastewater. Chemical pretreatments generated high concentrations of byproducts, mainly organic acids. No bacterial DNA was found in these pretreated biomasses. The acid pretreatment provided the highest carbohydrate solubilisation (98%) and monosaccharide recovery (81%). Enzymatic hydrolysis coupled with alkaline NaOH 2 M pretreatment achieved almost complete solubilisation of the biomass components, but high carbohydrate losses. Physical pretreatments remarkably increased the solubilisation of the biomass components during the enzymatic hydrolysis step, especially bead milling, which achieved solubilisation yields of 83% of carbohydrates, 43% of proteins, and 60% of lipids. The presence of viable bacteria in these pretreated biomasses could be related to the high carbohydrate losses and the generation of methanol and ethanol in addition to organic acids as byproducts.

Optimisation of the production of fermentable monosaccharides from algal biomass grown in photobioreactors treating wastewater.

Biomass grown in wastewater treatment photobioreactors is a cheap raw material with high contents of carbohydrates, proteins and lipids. This work studies the production of fermentable monosaccharides from three biomasses grown in piggery wastewater (P), domestic wastewater (W) and synthetic medium (S) by applying chemical pretreatment and enzymatic hydrolysis, using a Taguchi design. ANOVA identified temperature, chemical reagent type and chemical reagent concentration as significant operational parameters. However, the biomass concentration, pretreatment time, enzyme dosage and enzymatic hydrolysis time had no remarkable effect. The bacterial content of the biomass had no relevant impact on carbohydrate and protein solubilisation but had a remarkable effect on the degradation of the released carbohydrates (57, 60 and 37% for P, W and S), while also affecting lipid solubilisation. Pretreatment with HCl 2 M at 120 °C resulted the optimal conditions, achieving a monosaccharide recovery of 53, 59 and 80% for P, W and S biomasses, respectively.

Recovery of proteins from biomass grown in pig manure microalgae-based treatment plants by alkaline hydrolysis and acidic precipitation.

The influence of three variables on key parameters of the protein extraction process (an alkaline hydrolysis followed by an acidic precipitation) for biomass from innovative photo-bioreactors for pig manure treatment was evaluated. Alkaline hydrolysis provided high solubilisation values (up to 66.5% of the biomass), augmenting with increasing values of the three studied variables (NaOH concentration, temperature and time). Nevertheless, moderate total (13.2%) and protein extraction yields (16.9%) were obtained, which was attributable to protein denaturation or to the low effectivity of the precipitation method. Extracts rich in proteins (53.5%-77.9%) with suitable amino acid profiles were obtained, but significant amounts of the initial lipids (up to 44.6%) were co-extracted probably due to fatty acids saponification. These results establish the first step for future studies in enhancing cell wall disruption and protein recovery by coupling alkaline hydrolysis with other mechanical pre-treatments, while considering alternative separation and purification methods.

Study of steam explosion pretreatment and preservation methods of commercial cellulose.

Steam explosion (150-200 °C, 5-30 min) was performed on a commercial cellulose presented in two configurations (fiberized and compact sheet) and its effect on their chemical and physical properties was studied, along with the influence of two different preservation methods (acetone drying and freezing) after pretreatment. No degradation compounds were produced during pretreatment, although solid recovery (RS) decreased with temperature from 90% to 62%. Similar particle size and surface conditions (increased porosity) were found for both types of pretreated samples despite the extremely different initial configuration. Crystallinity diminished for 150 °C samples, but 200 °C pretreatment promoted recrystallization. Pretreatment also reduced polymerization degree, although enzymatic accessibility did not improve. Both acetone and freezing processes extremely affected cellulose properties. Acetone drying counterbalanced crystallinity and enzymatic accessibility variations of pretreated samples, while decreasing polymerization degree. Freezing dramatically decreased enzymatic accessibility of pretreated samples down to 15.8%.

Effect of pretreatments on biogas production from microalgae biomass grown in pig manure treatment plants.

Methane production from pretreated and raw mixed microalgae biomass grown in pig manure was evaluated. Acid and basic pretreatments provided the highest volatile solids solubilisation (up to 81%) followed by alkaline-peroxide and ultrasounds (23%). Bead milling and steam explosion remarkably increased the methane production rate, although the highest yield (377 mL CH4/g SV) was achieved by alkali pretreatment. Nevertheless, some pretreatments inhibited biogas production and resulted in lag phases of 7-9 days. Hence, experiments using only the pretreated solid phase were performed, which resulted in a decrease in the lag phase to 2-3 days for the alkali pretreatment and slightly increased biomass biodegradability of few samples. The limiting step during the BMP test (hydrolysis or microbial inhibition) for each pretreatment was elucidated using the goodness of fitting to a first order or a Gompertz model. Finally, the use of digestate as biofertilizer was evaluated applying a biorefinery concept.

Effect of ozonolysis parameters on the inhibitory compound generation and on the production of ethanol by Pichia stipitis and acetone-butanol-ethanol by Clostridium from ozonated and water washed sugarcane bagasse.

Sugarcane bagasse (SCB) was ozone pretreated and detoxified by water washing, applying a L9(3)(4) orthogonal array (OA) design of experiments to study the effect of pretreatment parameters (moisture content, ozone concentration, ozone/oxygen flow and particle size) on the generation of inhibitory compounds and on the composition of hydrolysates of ozonated-washed samples. Ozone concentration resulted the highest influence process parameter on delignification and sugar release after washing; while, for inhibitory compound formation, moisture content also had an important role. Ozone expended in pretreatment related directly with sugar release and inhibitory compound formation. Washing detoxification was effective, providing non-inhibitory hydrolysates. Maximum glucose and xylose release yields obtained were 84% and 67%, respectively, for ozonated-washed SCB. Sugar concentration resulted in the decisive factor for biofuels yields. Ethanol production achieved an 88% yield by Pichia stipitis, whereas Clostridium acetobutylicum produced 0.072gBUTANOL/gSUGAR and 0.188gABE/gSUGAR, and, Clostridium beijerinckii 0.165gBUTANOL/gSUGAR and 0.257gABE/gSUGAR.

Saccharification of microalgae biomass obtained from wastewater treatment by enzymatic hydrolysis. Effect of alkaline-peroxide pretreatment.

An enzymatic method for the carbohydrate hydrolysis of different microalgae biomass cultivated in domestic (DWB) and pig manure (PMWB) wastewaters, at different storage conditions (fresh, freeze-dried and reconstituted), was evaluated. The DWB provided sugars yields between 40 and 63%, although low xylose yields (< 23.5%). Approximately 2% of this biomass was converted to byproducts as succinic, acetic and formic acids. For PMWB, a high fraction of the sugars (up to 87%) was extracted, but mainly converted into acetic, butyric and formic acids, which was attributed to the bacterial action. In addition, the performance of an alkaline-peroxide pretreatment, conducted for 1h, 50°C and H2O2 concentrations from 1 to 7.5% (w/w), was essayed. The hydrolysis of pretreated microalgae supported a wide range of sugars extraction for DWB (55-90%), and 100% for PMWB. Nevertheless, a large fraction of these sugars (∼30% for DWB and 100% for PMWB) was transformed to byproducts.

Effect of ozonolysis pretreatment parameters on the sugar release, ozone consumption and ethanol production from sugarcane bagasse.

A L9(3)(4) orthogonal array (OA) experimental design was applied to study the four parameters considered most important in the ozonolysis pretreatment (moisture content, ozone concentration, ozone/oxygen flow and particle size) on ethanol production from sugarcane bagasse (SCB). Statistical analysis highlighted ozone concentration as the highest influence parameter on reaction time and sugars release after enzymatic hydrolysis. The increase on reaction time when decreasing the ozone/oxygen flow resulted in small differences of ozone consumptions. Design optimization for sugars release provided a parameters combination close to the best experimental run, where 77.55% and 56.95% of glucose and xylose yields were obtained, respectively. When optimizing the grams of sugar released by gram of ozone, the highest influence parameter was moisture content, with a maximum yield of 2.98gSUGARS/gO3. In experiments on hydrolysates fermentation, Saccharomyces cerevisiae provided ethanol yields around 80%, while Pichia stipitis was completely inhibited.

Saccharification of ozonated sugarcane bagasse using enzymes from Myceliophthora thermophila JCP 1-4 for sugars release and ethanol production.

The saccharification of ozonated sugarcane bagasse (SCB) by enzymes from Myceliophthora thermophila JCP 1-4 was studied. Fungal enzymes provided slightly higher sugar release than commercial enzymes, working at 50°C. Sugar release increased with temperature increase. Kinetic studies showed remarkable glucose release (4.99 g/L, 3%w/w dry matter) at 60°C, 8 h of hydrolysis, using an enzyme load of 10 FPU (filter paper unit). FPase and ß-glucosidase activities increased during saccharification (284% and 270%, respectively). No further significant improvement on glucose release was observed increasing the enzyme load above 7.5 FPU per g of cellulose. Higher dry matter contents increased sugars release, but not yields. The fermentation of hydrolysates by Saccharomyces cerevisiae provided glucose-to-ethanol conversions around to 63%.

Effect of thermal, acid, alkaline and alkaline-peroxide pretreatments on the biochemical methane potential and kinetics of the anaerobic digestion of wheat straw and sugarcane bagasse.

The effect of thermal, acid, alkaline and alkaline-peroxide pretreatments on the methane produced by the anaerobic digestion of wheat straw (WS) and sugarcane bagasse (SCB) was studied, using whole slurry and solid fraction. All the pretreatments released formic and acetic acids and phenolic compounds, while 5-hydroxymetilfurfural (HMF) and furfural were generated only by acid pretreatment. A remarkable inhibition was found in most of the whole slurry experiments, except in thermal pretreatment which improved methane production compared to the raw materials (29% for WS and 11% for SCB). The alkaline pretreatment increased biodegradability (around 30%) and methane production rate of the solid fraction of both pretreated substrates. Methane production results were fitted using first order or modified Gompertz equations, or a novel model combining both equations. The model parameters provided information about substrate availability, controlling step and inhibitory effect of compounds generated by each pretreatment.

Ozonolysis: An advantageous pretreatment for lignocellulosic biomass revisited.

Ozonolysis, as a lignocellulosic biomass pretreatment, goes back to 80s; however, in the last years it is becoming widespread again owing to its efficiency and mild operation conditions. Ozone reacts preferably with lignin than carbohydrates, promoting biomass destructuration and delignification, and so the sugar release by enzymatic hydrolysis. The hydrolysate from pretreated biomass has being used as sugars source for second-generation fuels production, mainly ethanol, methane and hydrogen. Short-chain carboxylic acids are the main inhibitory compounds generated, being properly removed by water washing. The most common inhibitory compounds reported for other pretreatments, furfural and HMF (5-hydroxymethylfurfural), are not found in ozone-pretreated hydrolysates. Composition of pretreated biomass and ozone consumption depends on several process parameters: reactor design, moisture content, particle size, pH, reaction time, ozone/air flow and ozone concentration. Additional studies are necessary to clarify process parameters effect and to optimize the process to achieve high yields with economic feasibility.

Application of pig slurry to soils. Effect of air stripping treatment on nitrogen and TOC leaching.

The effect of physical-chemical slurry treatment on the mobility and transformation of nitrogen and organic matter from pig slurry after soil application is evaluated. Two different pig slurries (one treated by stripping with air at pH=9 and another non-treated) were applied at the top of a soil column, containing approximately 100 kg of soil. Effluents were monitored measuring concentration values of ammonia, nitrites, nitrates and total organic carbon (TOC). The breakthrough curves were modelled using STANMOD and HYDRUS 1D codes. Low concentrations of ammonia were detected in the effluent recovered at the bottom of the soil profile for both types of slurry. Nitrate concentration in effluent was lower and more homogenous over time when applying stripping treated pig slurry. In N modelling, adsorption of ammonia by soil proved an important process, nitrite and nitrate adsorption being less significant, although not negligible. Transformation from ammonia to nitrite controls the kinetics of the nitrification process. Total organic carbon in the column effluent was higher in the experiment using treated pig slurry, which can be attributed to organic matter solubilisation in the stripping treatment process.