Recovery of polyhydroxyalkanoates (PHAs) from wastewater : a review

Polyhydroxyalkanoates (PHAs) are biopolyesters accumulated as carbon and energy storage materials under unbalanced growth conditions by various microorganisms. They are one of the most promising potential substitutes for conventional non-biodegradable plastics due to their similar physicochemical properties, but most important, its biodegradability. Production cost of PHAs is still a great barrier to extend its application at industrial scale. In order to reduce that cost, research is focusing on the use of several wastes as feedstock (such as agro-industrial and municipal organic waste and wastewater) in a platform based on mixed microbial cultures. This review provides a critical illustration of the state of the art of the most likely-to-be-scale-up PHA production processes using mixed microbial cultures platform and waste streams as feedstock, with a particular focus on both, upstream and downstream processes. Current pilot scale studies, future prospects, challenges and developments in the field are also highlighted

Polyhydroxyalkanoates production alongside wastewater treatment by mixed microbial cultures

El enfoque de este trabajo de investigación es analizar el proceso de producción de polihidroxialcanoatos (PHA) en paralelo al tratamiento de aguas residuales. Los PHA se consideran sustitutos potenciales de los plásticos convencionales y dentro del concepto de economía circular, son considerados como productos de valor añadido. Sin embargo, los altos costos durante la producción, han limitado un uso extensivo de los PHA. En los últimos años se han invertido esfuerzos en el desarrollo de alternativas de bajo costo que mejoren el rendimiento del proceso reduzcan los precios de obtención. El proceso de producción de PHA que se evalúa en esta tesis incluye las siguientes etapas: 1. Fermentación acidogénica: Durante esta etapa se estudió el potencial de diferentes residuos para producir ácidos grasos volátiles (AGV) mediante fermentación acidogénica. El uso de residuos de bajo costo como sustratos para la producción de PHA, supone una disminución importante en el costo general del proceso. En esta etapa, se analizó especialmente la composición de los AGV ya que es un parámetro clave para determinar tanto la composición del PHAA como su estructura y propiedades. 2. Etapa aerobia de enriquecimiento: durante esta etapa se llevó a cabo el enriquecimiento de un cultivo bacteriano mixto con microorganismos acumuladores de PHA. Los cultivos bacterianos mixtos son empleados como una alternativa económicamente viable para reemplazar el uso de cultivos puros. El proceso se operó en condiciones completamente aerobias con alternancia de presencia y ausencia de sustrato (festín/hambruna). En una primera fase se evaluó el efecto de trabajar a un pH controlado (pH = 7.5) o en ausencia de control de pH (en un intervalo de 8.8 a 9.2) empleando ácido acético como una única fuente de carbono. Posteriormente, se evaluó el enriquecimiento utilizando como sustrato un agua residual fermentada sintética de almazara. 3. Etapa de acumulación de PHA: el cultivo mixto enriquecido se empleó para realizar experimentos en discontinuos y evaluar efecto de diversos parámetros sobre el contenido final de PHA. El pH se evalúo en un rango de 4.0 a 0.9; además, se evaluó el efecto de la concentración de nutrientes (nitrógeno y fosforo) sobre el contenido máximo de PHAs. Se determinaron las condiciones óptimas para lograr una máxima acumulación de PHA tomando como base el perfil de oxígeno disuelto (OD). Así mismo, se evaluó la composición del PHA en función al uso de fuentes de carbono con diferentes composiciones. Se alcanzó una acumulación máxima de PHA del 73% gPHA g-1VSS empleando una mezcla 75:25 de ácido acético y ácido propiónico. 4. Extracción de PHA: para la extracción se utilizó dimetil carbonato (DMC) como “solvente verde” y la eficacia de recuperación se comparó con los resultados obtenidos al emplear cloroformo. A la par, se evaluó el efecto de emplear: i) un pre-tratamiento con hipoclorito de sodio (NaOCl) y ii) precipitación del biopolímero con etanol. Se logró una recuperación del 25% de PHA empleando el pretratamiento con NaOCl. El PHA recuperado se caracterizó en función a su composición, propiedades térmicas y peso molecular.The focus of this research work was conducted to analyse the three stages of a polyhydroxyalkanoates (PHA) production process alongside wastewater treatment. PHA are considered as potential substitutes of conventional plastics and the circular economy model, considers them as value-added products. However their production costs has limited their widespread, so currently many efforts are carried towards the development of cost-effective alternatives to enhance the PHA processes and reduce their costs. The process evaluated herein involves: 1. Acidogenic fermentation: Different wastes were evaluated in terms of their potential to produce volatile fatty acids (VFA) under acidogenic fermentation. Using wastes as non-expensive substrates for PHA production, can reduce the overall process costs. A lot of effort was carried on the determination of the VFA composition as it is a key parameter to determine the type of PHA synthesized and thus, their structure and properties. 2. Aerobic enrichment step: A mixed microbial culture (MMC) was used for the enrichment of PHA-accumulating microorganisms. MMC was used as an approach to reduce the costs related with the use of pure cultures. A sequencing batch reactor (SBR) was operated under feast/famine regimen. Firstly, acetic acid was used as sole carbon source and the effect over the PHA accumulation performance was studied by comparing the PHA content working at pH controlled (7.5) and without pH controlled. A second SBR was inoculated to enrich a MMC using a synthetic fermented olive mill waste water (OMW). 3. PHA accumulation: The enriched-MMC was used for batch experiments to promote high PHA contents. The influence of several parameters on the PHA maximum accumulation were evaluated. The pH influence was assessed in a range from 4.0 to 9.0. PHA accumulation under N and P limitation or excess was determined. The effect of the feeding strategy was also evaluated and the substrate composition influence on the final PHA composition and contents were elucidated. 4. Downstream processing: PHA extraction was done using DMC as green solvent and the recovery results were compared with the results obtained when chloroform is used. The idea of using DMC is to overcome the toxic environmental and human concerns related with the use of chlorinated solvents. The impact on the PHA recovery of applying a pre-tretament to boost cell lysis with sodium hypochlorite (NaOCl) or use a precipitation with methanol at the end of the process were investigated

Polyhydroxyalkanoates production alongside wastewater treatment by mixed microbial cultures

El enfoque de este trabajo de investigación es analizar el proceso de producción de polihidroxialcanoatos (PHA) en paralelo al tratamiento de aguas residuales. Los PHA se consideran sustitutos potenciales de los plásticos convencionales y dentro del concepto de economía circular, son considerados como productos de valor añadido. Sin embargo, los altos costos durante la producción, han limitado un uso extensivo de los PHA. En los últimos años se han invertido esfuerzos en el desarrollo de alternativas de bajo costo que mejoren el rendimiento del proceso reduzcan los precios de obtención. El proceso de producción de PHA que se evalúa en esta tesis incluye las siguientes etapas: 1. Fermentación acidogénica: Durante esta etapa se estudió el potencial de diferentes residuos para producir ácidos grasos volátiles (AGV) mediante fermentación acidogénica. El uso de residuos de bajo costo como sustratos para la producción de PHA, supone una disminución importante en el costo general del proceso. En esta etapa, se analizó especialmente la composición de los AGV ya que es un parámetro clave para determinar tanto la composición del PHAA como su estructura y propiedades. 2. Etapa aerobia de enriquecimiento: durante esta etapa se llevó a cabo el enriquecimiento de un cultivo bacteriano mixto con microorganismos acumuladores de PHA. Los cultivos bacterianos mixtos son empleados como una alternativa económicamente viable para reemplazar el uso de cultivos puros. El proceso se operó en condiciones completamente aerobias con alternancia de presencia y ausencia de sustrato (festín/hambruna). En una primera fase se evaluó el efecto de trabajar a un pH controlado (pH = 7.5) o en ausencia de control de pH (en un intervalo de 8.8 a 9.2) empleando ácido acético como una única fuente de carbono. Posteriormente, se evaluó el enriquecimiento utilizando como sustrato un agua residual fermentada sintética de almazara. 3. Etapa de acumulación de PHA: el cultivo mixto enriquecido se empleó para realizar experimentos en discontinuos y evaluar efecto de diversos parámetros sobre el contenido final de PHA. El pH se evalúo en un rango de 4.0 a 0.9; además, se evaluó el efecto de la concentración de nutrientes (nitrógeno y fosforo) sobre el contenido máximo de PHAs. Se determinaron las condiciones óptimas para lograr una máxima acumulación de PHA tomando como base el perfil de oxígeno disuelto (OD). Así mismo, se evaluó la composición del PHA en función al uso de fuentes de carbono con diferentes composiciones. Se alcanzó una acumulación máxima de PHA del 73% gPHA g-1VSS empleando una mezcla 75:25 de ácido acético y ácido propiónico. 4. Extracción de PHA: para la extracción se utilizó dimetil carbonato (DMC) como “solvente verde” y la eficacia de recuperación se comparó con los resultados obtenidos al emplear cloroformo. A la par, se evaluó el efecto de emplear: i) un pre-tratamiento con hipoclorito de sodio (NaOCl) y ii) precipitación del biopolímero con etanol. Se logró una recuperación del 25% de PHA empleando el pretratamiento con NaOCl. El PHA recuperado se caracterizó en función a su composición, propiedades térmicas y peso molecular.The focus of this research work was conducted to analyse the three stages of a polyhydroxyalkanoates (PHA) production process alongside wastewater treatment. PHA are considered as potential substitutes of conventional plastics and the circular economy model, considers them as value-added products. However their production costs has limited their widespread, so currently many efforts are carried towards the development of cost-effective alternatives to enhance the PHA processes and reduce their costs. The process evaluated herein involves: 1. Acidogenic fermentation: Different wastes were evaluated in terms of their potential to produce volatile fatty acids (VFA) under acidogenic fermentation. Using wastes as non-expensive substrates for PHA production, can reduce the overall process costs. A lot of effort was carried on the determination of the VFA composition as it is a key parameter to determine the type of PHA synthesized and thus, their structure and properties. 2. Aerobic enrichment step: A mixed microbial culture (MMC) was used for the enrichment of PHA-accumulating microorganisms. MMC was used as an approach to reduce the costs related with the use of pure cultures. A sequencing batch reactor (SBR) was operated under feast/famine regimen. Firstly, acetic acid was used as sole carbon source and the effect over the PHA accumulation performance was studied by comparing the PHA content working at pH controlled (7.5) and without pH controlled. A second SBR was inoculated to enrich a MMC using a synthetic fermented olive mill waste water (OMW). 3. PHA accumulation: The enriched-MMC was used for batch experiments to promote high PHA contents. The influence of several parameters on the PHA maximum accumulation were evaluated. The pH influence was assessed in a range from 4.0 to 9.0. PHA accumulation under N and P limitation or excess was determined. The effect of the feeding strategy was also evaluated and the substrate composition influence on the final PHA composition and contents were elucidated. 4. Downstream processing: PHA extraction was done using DMC as green solvent and the recovery results were compared with the results obtained when chloroform is used. The idea of using DMC is to overcome the toxic environmental and human concerns related with the use of chlorinated solvents. The impact on the PHA recovery of applying a pre-tretament to boost cell lysis with sodium hypochlorite (NaOCl) or use a precipitation with methanol at the end of the process were investigated

Polyhydroxyalkanoates production alongside wastewater treatment by mixed microbial cultures /

El enfoque de este trabajo de investigación es analizar el proceso de producción de polihidroxialcanoatos (PHA) en paralelo al tratamiento de aguas residuales. Los PHA se consideran sustitutos potenciales de los plásticos convencionales y dentro del concepto de economía circular, son considerados como productos de valor añadido. Sin embargo, los altos costos durante la producción, han limitado un uso extensivo de los PHA. En los últimos años se han invertido esfuerzos en el desarrollo de alternativas de bajo costo que mejoren el rendimiento del proceso reduzcan los precios de obtención. El proceso de producción de PHA que se evalúa en esta tesis incluye las siguientes etapas: 1. Fermentación acidogénica: Durante esta etapa se estudió el potencial de diferentes residuos para producir ácidos grasos volátiles (AGV) mediante fermentación acidogénica. El uso de residuos de bajo costo como sustratos para la producción de PHA, supone una disminución importante en el costo general del proceso. En esta etapa, se analizó especialmente la composición de los AGV ya que es un parámetro clave para determinar tanto la composición del PH19 como su estructura y propiedades. 2. Etapa aerobia de enriquecimiento: durante esta etapa se llevó a cabo el enriquecimiento de un cultivo bacteriano mixto con microorganismos acumuladores de PHA. Los cultivos bacterianos mixtos son empleados como una alternativa económicamente viable para reemplazar el uso de cultivos puros. El proceso se operó en condiciones completamente aerobias con alternancia de presencia y ausencia de sustrato (festín/hambruna). En una primera fase se evaluó el efecto de trabajar a un pH controlado (pH = 7.5) o en ausencia de control de pH (en un intervalo de 8.8 a 9.2) empleando ácido acético como una única fuente de carbono. Posteriormente, se evaluó el enriquecimiento utilizando como sustrato un agua residual fermentada sintética de almazara. 3. Etapa de acumulación de PHA: el cultivo mixto enriquecido se empleó para realizar experimentos en discontinuos y evaluar efecto de diversos parámetros sobre el contenido final de PHA. El pH se evalúo en un rango de 4.0 a 0.9; además, se evaluó el efecto de la concentración de nutrientes (nitrógeno y fosforo) sobre el contenido máximo de PHAs. Se determinaron las condiciones óptimas para lograr una máxima acumulación de PHA tomando como base el perfil de oxígeno disuelto (OD). Así mismo, se evaluó la composición del PHA en función al uso de fuentes de carbono con diferentes composiciones. Se alcanzó una acumulación máxima de PHA del 73% gPHA g-1VSS empleando una mezcla 75:25 de ácido acético y ácido propiónico. 4. Extracción de PHA: para la extracción se utilizó dimetil carbonato (DMC) como "solvente verde" y la eficacia de recuperación se comparó con los resultados obtenidos al emplear cloroformo. A la par, se evaluó el efecto de emplear: i) un pre-tratamiento con hipoclorito de sodio (NaOCl) y ii) precipitación del biopolímero con etanol. Se logró una recuperación del 25% de PHA empleando el pretratamiento con NaOCl. El PHA recuperado se caracterizó en función a su composición, propiedades térmicas y peso molecular.The focus of this research work was conducted to analyse the three stages of a polyhydroxyalkanoates (PHA) production process alongside wastewater treatment. PHA are considered as potential substitutes of conventional plastics and the circular economy model, considers them as value-added products. However their production costs has limited their widespread, so currently many efforts are carried towards the development of cost-effective alternatives to enhance the PHA processes and reduce their costs. The process evaluated herein involves: 1. Acidogenic fermentation: Different wastes were evaluated in terms of their potential to produce volatile fatty acids (VFA) under acidogenic fermentation. Using wastes as non-expensive substrates for PHA production, can reduce the overall process costs. A lot of effort was carried on the determination of the VFA composition as it is a key parameter to determine the type of PHA synthesized and thus, their structure and properties. 2. Aerobic enrichment step: A mixed microbial culture (MMC) was used for the enrichment of PHA-accumulating microorganisms. MMC was used as an approach to reduce the costs related with the use of pure cultures. A sequencing batch reactor (SBR) was operated under feast/famine regimen. Firstly, acetic acid was used as sole carbon source and the effect over the PHA accumulation performance was studied by comparing the PHA content working at pH controlled (7.5) and without pH controlled. A second SBR was inoculated to enrich a MMC using a synthetic fermented olive mill waste water (OMW). 3. PHA accumulation: The enriched-MMC was used for batch experiments to promote high PHA contents. The influence of several parameters on the PHA maximum accumulation were evaluated. The pH influence was assessed in a range from 4.0 to 9.0. PHA accumulation under N and P limitation or excess was determined. The effect of the feeding strategy was also evaluated and the substrate composition influence on the final PHA composition and contents were elucidated. 4. Downstream processing: PHA extraction was done using DMC as green solvent and the recovery results were compared with the results obtained when chloroform is used. The idea of using DMC is to overcome the toxic environmental and human concerns related with the use of chlorinated solvents. The impact on the PHA recovery of applying a pre-tretament to boost cell lysis with sodium hypochlorite (NaOCl) or use a precipitation with methanol at the end of the process were investigated

Phosphorus recovery from mixed microbial culture: production and extraction through green methods

Large scale polyhydroxyalkanoates (PHA) production is limited by high production costs compared to that of petroleum-based plastics. Two key factors can be pin down to reduce costs: i) the use of mixed microbial cultures (MMC) instead of pure cultures and ii) the application of cheap and environmental friendly recovery technologies. In this work, the MMC biomass with PHA-accumulating capacity was selected in a sequencing batch reactor fed with a synthetic effluent emulating a fermented oil mill wastewater. The biomass was harvested and transferred to an accumulation reactor, where PHA contents up to 54% of the dry cell weight were obtained, using a mixture of acetic and propionic acids. A copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate with 47% (wt) 3-hydroxyvalerate was obtained. The polymer extraction was done in aqueous phase using chemicals that destroy the non-PHA cellular material releasing the polymer’s granules. The effectiveness of the switchable anionic surfactant NH4-Laurate was investigated and compared with that of sodium dodecyl sulphate and NH4OH by testing them on lyophilized biomass, with and without a NaClO pre-treatment. Further, a purification post-treatment was performed on some of the samples as well. When operating the extraction at 90°C for 3h with a ratio surfactant to biomass of 200:100 w/w, gas chromatography analysis showed that all the tested extraction agents allow obtaining PHA with an excellent purity (≈100%) after a pre-treatment with NaClO at 100°C for 1 h. The highest recovery yield (73 %) was obtained when using NH4-Laurate for which operating conditions of the extraction process such as temperature, concentration and contact time have been optimized. When the extracted polymer was washed with a 0.1 N NH4OH solution and ethanol, purity was improved, but lower recovery yields were obtained. To determine the effect of extraction process on the PHA purity, Nuclear Magnetic Resonance Spectroscopy was used

Improvement of the Polyhydroxyalkanoates Recovery from Mixed Microbial Cultures Using Sodium Hypochlorite Pre-Treatment Coupled with Solvent Extraction

The use of mixed microbial cultures (MMC) and organic wastes and wastewaters as feed sources is considered an appealing approach to reduce the current polyhydroxyalkanoates (PHAs) production costs. However, this method entails an additional hurdle to the PHAs downstream processing (recovery and purification). In the current work, the effect of a sodium hypochlorite (NaClO) pre-treatment coupled with dimethyl carbonate (DMC) or chloroform (CF) as extraction solvents on the PHAs recovery efficiency (RE) from MMC was evaluated. MMC were harvested from a sequencing batch reactor (SBR) fed with a synthetic prefermented olive mill wastewaster. Two different carbon-sources (acetic acid and acetic/propionic acids) were employed during the batch accumulation of polyhydroxybutyrate (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from MMC. Obtained PHAs were characterized by 1H and 13C nuclear magnetic resonance, gel-permeation chromatography, differential scanning calorimetry, and thermal gravimetric analysis. The results showed that when a NaClO pre-treatment is not added, the use of DMC allows to obtain higher RE of both biopolymers (PHB and PHBV), in comparison with CF. In contrast, the use of CF as extraction solvent required a pre-treatment step to improve the PHB and PHBV recovery. In all cases, RE values were higher for PHBV than for PHB