Periodic Reporting for period 1 - AlgalBB (Development of Cost-Effective Process for Phyco-Remediation of Dairy Wastewater and Valorization of Algal Biomass for Production of Biofuel and Biochemical: A Sustainable Approach towards Bio-Refinery)
Reporting period: 2023-05-22 to 2025-05-21
Currently, Dairy Wastewater (DW) is seen as a major global problem, difficult and expensive to treat and potentially very harmful to the environment as it contains high amounts of organic and inorganic content. The current treatment methods (physicochemical and conventional) are energy-intensive and don’t sufficiently remove the nutrients. But, with the high amount that is produced worldwide: 0.2‒10 L wastewater/ L of milk processed; this is potentially a valuable resource that, if utilized appropriately, can provide high-value green chemicals and biofuel. Therefore, a new cost-effective method for the treatment of DW along with the generation of valuable biomass needs to be developed.
In this context, AlgalBB proposes the concept of biorefinery for the substantive treatment of DW via microalgae, coupled with biofuel and bio-based chemical production. The objectives have been pursued through:
1) The designing of a microalgae consortia that can remediate DW and generate lipid/carbohydrate-rich algal biomass,
2) Development of multienzyme magnetic nanocatalyst for efficient enzymatic hydrolysis to recover fermentable sugars from algal biomass,
3) Immobilization of fermentative bacteria for fermentation of sugars into bio-based organic acids,
4) Development of in-situ transesterification process for wet algal biomass to produce biofuel.
In this context, AlgalBB proposes the concept of biorefinery for the substantive treatment of DW via microalgae, coupled with biofuel and bio-based chemical production. The objectives have been pursued through:
1) The designing of a microalgae consortia that can remediate DW and generate lipid/carbohydrate-rich algal biomass,
2) Development of multienzyme magnetic nanocatalyst for efficient enzymatic hydrolysis to recover fermentable sugars from algal biomass,
3) Immobilization of fermentative bacteria for fermentation of sugars into bio-based organic acids,
4) Development of in-situ transesterification process for wet algal biomass to produce biofuel.
1) For the designing of microalgae consortia, the current project assessed different combinations of non-flocculating microalgae species (C. vulgaris and Scenedesmus sp) with flocculating filamentous microalgae species (Tribonema and Lyngbya sp.). For the monoculture study, all four microalgae cultures were inoculated into the wastewater with an initial concentration of 0.1 g/ L . For the consortia study, 9 combinations of microalgae sp. were tested at the ratio of 1:1 for two species, 1:1:1 for three species, and 1:1:1:1 for four species with the initial concentration of microalgae 0.1 g/L in all runs. The present study conducted a total of 13 runs for 15 days. The study also tested the effect of harvesting time on biochemical compositions of monoculture microalgae sp. and microalgae consortia by withdrawing the culture on days 7, 9, 11, and 15. Key parameters, including organic, inorganic nutrient removal efficiency, biomass productivity, and the biochemical component (carbohydrate, lipid, protein), have been systematically analyzed to assess the effectiveness of the co-cultivation system. Further, the harvesting potential of microalgae consortia, at the end day, i.e. day 15, was checked by withdrawing the sample at different time intervals, i.e. 0, 1, 2, and 3 h.
Achievement: Successfully designed microalgae consortia (Scenedesmus and Tribonema) that removed >85% pollutants from DW and accumulated higher carbohydrate > 50%; Achieved harvesting efficiency of algal biomass >75%.
2) A multi-enzyme magnetic nanocatalyst (ME-MNC), comprising cellulase, α-amylase, amyloglucosidase, and alcalase, was developed for one-pot hydrolysis of microalgae consortia (Scenedesmus and Tribonema) cultivated in dairy wastewater. Enzyme immobilization onto amino-functionalized iron oxide nanoparticles was optimized using Central Composite Design to evaluate the combined effects of glutaraldehyde concentration, crosslinking time, and immobilization time on activity recovery of the four enzymes. The performance of the ME-MNC has been assessed using microalgae consortia cultivated in DW. A series of experiments have been conducted to determine the optimal conditions for hydrolysis by varying pH (3–8) and temperature (30–70 °C), aiming to achieve maximum yields of reducing sugars and protein under specific conditions. Further, the reusability of the ME-MNC for biomass saccharification has been evaluated in up to five batch processes until a notable decrease in soluble sugar yield is detected.
Achievement: The optimal conditions were determined as 103.82 mM glutaraldehyde concentration, 178.79 min of crosslinking time, and 7.08 h of immobilization time. Experimental enzyme activity recovery closely matched predicted values, achieving 64.66% for cellulase, 67.02% for α-amylase, 43.4% for amyloglucosidase, and 81.14% for alcalase. Characterization of ME-MNC using X-ray diffraction, Fourier transform infrared spectrophotometry, field-emission scanning electron microscopy and thermogravimetric analysis confirmed successful synthesis. The ME-MNC achieved 81.72% sugar recovery, surpassing the 69.5% yield of the free enzyme mixture, and demonstrated stability across a broad pH range (6–8) and temperature range (50–60°C). Additionally, the ME-MNC retained 81% residual sugar recovery and 96% protein recovery after five reuse cycles, underscoring its high efficiency and reusability.
3) A protocol for immobilization of Actinobacillus succinogenes on calcium alginate–coated iron oxide nanoparticles was developed. The immobilized bacteria were tested on five different synthetic fermentation media, and the most efficient medium was selected for evaluating the reusability of the immobilized bacteria. Initially, fermentation was conducted using a synthetic medium (sugar composition was similar to microalgal hydrolysate), and after optimization, synthetic sugars were replaced with microalgal hydrolysate to assess the production of organic acids. This transition enabled the evaluation of the fermentation process using a renewable and sustainable feedstock derived from DW.
Achievement: The fermentation process in a synthetic medium using immobilized Actinobacillus succinogenes resulted in the production of lactic acid, with a maximum yield of 8.68 g/L and a maximum productivity of 0.21 g/L/h. Additionally, the immobilized bacteria were successfully reused for five cycles without any observed decrease in lactic acid production.
4) Transesterification was successfully initiated using microalgal consortia biomass under experimental conditions involving methanol, sulfuric acid, and, dichloromethane (DCM), and a reaction temperature of 50 °C over 96 hours.
Achievement: The reaction led to the formation of free fatty acids (FFAs), indicating successful lipid breakdown. However, biodiesel (FAME) yield was minimal, likely due to hydrolysis driven by residual water and the acidic environment.
Achievement: Successfully designed microalgae consortia (Scenedesmus and Tribonema) that removed >85% pollutants from DW and accumulated higher carbohydrate > 50%; Achieved harvesting efficiency of algal biomass >75%.
2) A multi-enzyme magnetic nanocatalyst (ME-MNC), comprising cellulase, α-amylase, amyloglucosidase, and alcalase, was developed for one-pot hydrolysis of microalgae consortia (Scenedesmus and Tribonema) cultivated in dairy wastewater. Enzyme immobilization onto amino-functionalized iron oxide nanoparticles was optimized using Central Composite Design to evaluate the combined effects of glutaraldehyde concentration, crosslinking time, and immobilization time on activity recovery of the four enzymes. The performance of the ME-MNC has been assessed using microalgae consortia cultivated in DW. A series of experiments have been conducted to determine the optimal conditions for hydrolysis by varying pH (3–8) and temperature (30–70 °C), aiming to achieve maximum yields of reducing sugars and protein under specific conditions. Further, the reusability of the ME-MNC for biomass saccharification has been evaluated in up to five batch processes until a notable decrease in soluble sugar yield is detected.
Achievement: The optimal conditions were determined as 103.82 mM glutaraldehyde concentration, 178.79 min of crosslinking time, and 7.08 h of immobilization time. Experimental enzyme activity recovery closely matched predicted values, achieving 64.66% for cellulase, 67.02% for α-amylase, 43.4% for amyloglucosidase, and 81.14% for alcalase. Characterization of ME-MNC using X-ray diffraction, Fourier transform infrared spectrophotometry, field-emission scanning electron microscopy and thermogravimetric analysis confirmed successful synthesis. The ME-MNC achieved 81.72% sugar recovery, surpassing the 69.5% yield of the free enzyme mixture, and demonstrated stability across a broad pH range (6–8) and temperature range (50–60°C). Additionally, the ME-MNC retained 81% residual sugar recovery and 96% protein recovery after five reuse cycles, underscoring its high efficiency and reusability.
3) A protocol for immobilization of Actinobacillus succinogenes on calcium alginate–coated iron oxide nanoparticles was developed. The immobilized bacteria were tested on five different synthetic fermentation media, and the most efficient medium was selected for evaluating the reusability of the immobilized bacteria. Initially, fermentation was conducted using a synthetic medium (sugar composition was similar to microalgal hydrolysate), and after optimization, synthetic sugars were replaced with microalgal hydrolysate to assess the production of organic acids. This transition enabled the evaluation of the fermentation process using a renewable and sustainable feedstock derived from DW.
Achievement: The fermentation process in a synthetic medium using immobilized Actinobacillus succinogenes resulted in the production of lactic acid, with a maximum yield of 8.68 g/L and a maximum productivity of 0.21 g/L/h. Additionally, the immobilized bacteria were successfully reused for five cycles without any observed decrease in lactic acid production.
4) Transesterification was successfully initiated using microalgal consortia biomass under experimental conditions involving methanol, sulfuric acid, and, dichloromethane (DCM), and a reaction temperature of 50 °C over 96 hours.
Achievement: The reaction led to the formation of free fatty acids (FFAs), indicating successful lipid breakdown. However, biodiesel (FAME) yield was minimal, likely due to hydrolysis driven by residual water and the acidic environment.
This study advances microalgae-based wastewater remediation and biomass valorization by developing a high-efficiency Scenedesmus-Tribonema (S:T) consortium, achieving 12–174% higher biomass productivity and superior dairy wastewater treatment (86.7% COD, >88.7% NO₃⁻-N, and >98.5% PO₄³⁻-P removal) compared to monocultures. Optimized harvesting strategies maximized carbohydrate (54.84%) and lipid recovery (9.87%), while the novel multi-enzyme magnetic nanocatalyst (ME-MNC) significantly improved biomass hydrolysis efficiency (81.72% sugar recovery, stable across pH 6–8 and 50–60°C, retaining 81% residual sugar after five reuse cycles). Additionally, the immobilization of Actinobacillus succinogenes using calcium alginate-coated iron oxide nanoparticles enhanced fermentation stability and efficiency by 178% compared to free cells, and demonstrated reusability for up to five cycles. To ensure further uptake and success, pilot-scale validation, and techno-economic analysis can be performed.