Development of a microalgae-based consortium for the bioremediation of sugar mill effluent

Abstract

Industrial and agricultural activities have increased exponentially to meet the rising demands for food. The sugar production process is water-intensive and requires high volumes of freshwater that are subsequently discharged as effluent. An average of 1000 L of wastewater is produced per ton of sugarcane processed. The sugarcane industry wastewater is characterized by high chemical oxygen demand (COD: 1752 - 8339 mg L-1), and biochemical oxygen demand (BOD:1052 - 4641 mg L-1) but remains low in nutrients and other minerals. Discharging untreated wastewater into the environment might have negative consequences, thus reusing and treating wastewater is essential. Conventional physiochemical treatment methods including sedimentation, filtration, and coagulation-flocculation have shown limited efficacy. The sugar industry wastewater exhibits high biodegradability, thus biological treatment techniques are preferred due to environmental friendliness and sustainability. In recent years, the co-culturing approach has gained interest as a strategy to improve the biotechnological productivity of microalgae in wastewater treatment. This study aims to develop stable microalgae-based consortia using native, microalgal, bacterial and yeast strains. The steps adopted for achieving the above-stated aim were: (1) isolation, identification, and characterization of indigenous microorganisms from wastewater, and (2) assembling microbial consortia to identify the most effective and compatible strains. Indigenous microalgal, bacterial, and yeast strains were isolated and screened for growth in synthetic wastewater. The strains exhibiting significant growth were further characterized in real wastewater from the sugar industry to evaluate their performance efficiency based on COD removal efficiencies. To attain higher wastewater treatment efficiencies, different primary and secondary combinations of consortia were constructed from the pool of previously screened and selected microbial strains guided by the bottom-up approach. The microalgal, bacterial, and yeast microbial strains present in the final consortia were identified using polymerase chain reaction and sequencing. The final microalgal-based consortia were characterised in real effluent to assess wastewater treatment efficiency and elucidate their basic interactions for better application. Two microalgal, seven bacterial, and four yeast strains were isolated from the sugar industry wastewater. In the primary screening procedure in synthetic wastewater, two microalgal strains (A7 and C12), and four bacterial (B003, B009, B010, and B013) strains were found to grow substantially and thus selected for further studies and subsequent microalgae-consortia development. The two microalgal strains showed high removal efficiency for NO3--N (98–100%), NH4+-N (62-65%), and PO43--P (75-80%), however, the removal rate for COD was mainly observed in bacterial strains ranging between 1–73%. Also, the yeast strain (Y2) reduced COD from 22660 to 11690 mg L-1 (48% removal rate) within 168 h of cultivation. The co-culturing of microalgae with bacteria and yeast could improve the treatment of high-strength wastewater. In this regard, four primary, and three secondary consortia were considered. In all the primary consortia (B009A7, B010A7, B013A7, and Y2A7), improvement in removal efficiency for Total Nitrogen (TN) and Total phosphorus (TP) was recorded in ranges between 75-80% and 84-94%, respectively. However, a significantly lower removal rate (4-7%) was observed for COD. Furthermore, three secondary (B009B010A7, B009B013A7, and B010B013A7) and one tertiary consortia (B009B010B013A7) were considered. All secondary consortia exhibited a prolonged lag phase with reduced COD removal efficiency. In contrast, the tertiary consortia showed improved COD, TN, and TP removal efficiency corresponding to 26%, 85% & 73% (respectively) in synthetic wastewater. The microalgal, bacterial, and yeast strains in the final consortia were identified as Chlorella sorokiniana A7, Rhodococcus sp B009, Bacillus sp B010, Bacillus sp B013, and Saccharomyces cerevisiae Y2. The wastewater treatment efficiency of the consortia (MBC and MYC) was further evaluated in real sugar industry wastewater. The COD removal efficiency was found to be 86% and 71% after 4 days of cultivation for MBC and MYC, respectively. In addition, the co-culture with S. cerevisiae Y2 markedly increased chlorophyll-a content, photosynthesis, and respiration in microalgae. The microalgal-based consortia exhibited physical and biochemical interactions, with improved yield parameters and metabolite production between microalgae, bacteria, and yeast. The co-cultivation of Chlorella sorokiniana A7 and Saccharomyces cerevisiae Y2 was observed to have the highest COD removal efficiency from wastewater (100% within 4 d of cultivation). Microalgae and yeast mutually benefited from each other in the MYC system with synergistic cross-feeding between specific parameters such as CO2/O2, and organic acids. In addition, indole-3-acetic acid (IAA) was selected as a marker for evaluating the plant growth-promoting effects of co-cultured partners and determining the communication intensity. All co-cultured strains were found to produce and secrete indole acetic acid (IAA), suggesting plant growth-promoting effects. All the co-culture partners produced different concentrations of IAA under tryptophan ranging between 2 to 129 mg L-1. Meanwhile, IAA production was highest within 24 hr of cultivation in the MBC system, while the MYC exhibited a steady increase in IAA production, with the highest production observed after 72 h. The IAA signals are suggested to facilitate the establishment of mutualistic associations between microalgae and yeast/bacteria under varying environmental conditions. This indicates that yeast/bacteria may promote the growth of the co-existing microalgae through secretion of IAA, and microalgae would selectively enhance IAA secretion, thus, shaping the physiology and ecology of the partners in the microbial consortia. The study demonstrated the efficacy of microalgae-based consortia that have potential use in treating high-strength COD wastewater. The results could help improve the performance of the current treatment methods by introducing low-cost and sustainable biological technologies. The study demonstrated that microalgal-based co-cltivation is a promising bioremediation tool for high-strength biodegradable wastewater and presents environmental value in the design of low-energy, small-scale biological treatment systems. An insight into mechanisms of interactions between microalgae and co-cultured microbes still requires further study through integrated omics, studying the ecology and diversity of microbial communities could improve their application in environmental monitoring and bioremediation.