Antagonistic properties of Lactiplantibacillus plantarum MYSVB1 against Alternaria alternata: a putative probiotic strain isolated from the banyan tree fruit.

Alternaria alternata, a notorious phytopathogenic fungus, has been documented to infect several plant species, leading to the loss of agricultural commodities and resulting in significant economic losses. Lactic acid bacteria (LAB) hold immense promise as biocontrol candidates. However, the potential of LABs derived from fruits remains largely unexplored. In this study, several LABs were isolated from tropical fruit and assessed for their probiotic and antifungal properties. A total of fifty-five LABs were successfully isolated from seven distinct fruits. Among these, seven isolates showed inhibition to growth of A. alternata. Two strains, isolated from fruits: Ficus benghalensis, and Tinospora cordifolia exhibited promising antifungal properties against A. alternata. Molecular identification confirmed their identities as Lactiplantibacillus plantarum MYSVB1 and MYSVA7, respectively. Both strains showed adaptability to a wide temperature range (10-45°C), and salt concentrations (up to 7%), with optimal growth around 37 °C and high survival rates under simulated gastrointestinal conditions. Among these two strains, Lpb. plantarum MYSVB1 demonstrated significant inhibition (p < 0.01) of the growth of A. alternata. The inhibitory effects of cell-free supernatant (CFS) were strong, with 5% crude CFS sufficient to reduce fungal growth by >70% and complete inhibition by 10% CFS. Moreover, the CFS was inhibitory for both mycelial growth and conidial germination. CFS retained its activity even after long cold storage. The chromatographic analysis identified organic acids in CFS, with succinic acid as the predominant constituent, with lactic acid, and malic acid in descending order. LAB strains isolated from tropical fruits showed promising probiotic and antifungal properties, making them potential candidates for various applications in food and agriculture.

Pilot-scale aerobic granular sludge reactors with granular activated carbon for effective nitrogen and phosphorus removal from domestic wastewater.

Aerobic granular sludge (AGS) is a breakthrough biotechnology of 21st century and an innovative alternative to activated sludge for treating wastewater. Concerns on long-start up periods for development of AGS and stability of granules are impeding its widespread implementation for treating low-strength domestic wastewater especially in tropical climate conditions. Addition of nucleating agents have been shown to improve development of AGS while treating low-strength wastewaters. There are no previous studies on AGS development and biological nutrient removal (BNR) in the presence of nucleating agents during treatment of real domestic wastewater. This study investigated AGS formation and BNR pathways while treating real domestic wastewater in a 2 m3 pilot-scale granular sequencing batch reactor (gSBR) operated without and with granular activated carbon (GAC) particles. The gSBRs were operated under tropical climate (T ≈ 30 °C) for >4-years to evaluate the effect of GAC addition on granulation, granular stability and BNR at pilot-scale. Formation of granules was observed within 3 months. MLSS values of 4 and 8 g/L were recorded within 6 months in gSBRs without and with GAC particles, respectively. The granules had an average size of 1.2 mm and SVI5 of 22 mL/g. Ammonium was mainly removed through nitrate formation in the gSBR without GAC. But, ammonium was removed by short-cut nitrification via nitrite due to washout of nitrite oxidizing bacteria in the presence of GAC. Phosphorus removal was much higher in gSBR with GAC due to the establishment of enhanced biological phosphorus removal (EBPR) pathway. After 3 months, the phosphorus removal efficiencies were at 15 % and 75 %, respectively, without and with GAC particles. The addition of GAC led to moderation in bacterial community and enrichment of polyphosphate-accumulating organisms. This is the first ever report on pilot-scale demonstration of AGS technology in the Indian sub-continent and GAC addition on BNR pathways.

De novo granulation of sewage-borne microorganisms: A proof of concept on cultivating aerobic granular sludge without activated sludge and effective enhanced biological phosphorus removal.

Long start-up periods for granulating activated sludge and concerns on granular stability are the bottlenecks reported during implementation of novel aerobic granular sludge (AGS) technology in municipal wastewater treatment plants. Here, de novo granulation of sewage-borne microorganisms without using activated sludge (AS) inoculum was investigated in bench-scale sequencing batch reactors (SBR). Data showed that formation of AGS from sewage-borne microorganisms was rapid and first granules appeared within one week. Granulation was indicated by appearance of biomass particles (size >0.12 mm), high biomass levels (∼8 g/L) and superior settling properties (SVI30 min: 30 mL/g). Granulation process involved distinct stages like formation of aggregates, retention of aggregates, and growth of millimetre sized granules. Simultaneous COD, nitrogen and phosphorous removal was established within 10 days of start-up in the SBR without using AS inoculum. However, phosphorus removal became stable after 50 days of start-up. Total nitrogen (TN) and total phosphorus (TP) removals of 92% and 70%, respectively, were achieved from real domestic wastewater. Furthermore, addition of granular activated carbon (GAC) had improved both granulation and biological nutrient removals. Interestingly, phosphorus removal became quite stable within 10 days of start-up in the SBR operated with GAC particles. TN and TP removals were found to be higher at >98% and >94%, respectively, in GAC-augmented SBR. Removal of ammonia and phosphorus were mediated by nitritation-denitritation and enhanced biological phosphorus removal (EBPR) pathways, respectively. The bacterial diversity of AGS was lower than that of sewage. Quantitative PCR indicated enrichment of ammonia oxidizing bacteria, denitrifying bacteria and polyphosphate accumulating organisms during granulation. De novo granulation of sewage-borne microorganisms is a promising approach for rapidly cultivating AGS and establishing biological nutrient removal in sewage treatment plants.

Selenite reduction and biogenesis of selenium-nanoparticles by different size groups of aerobic granular sludge under aerobic conditions.

Microbial transformations play a vital role in Se cycle in the environment and decrease the solubility and toxicity of Se oxyanions by converting to elemental selenium (Se0) nanostructures. Aerobic granular sludge (AGS) has attracted interest due to efficient reduction of selenite to biogenic Se0 (Bio-Se0) and retention in bioreactors. Here, selenite removal, biogenesis of Bio-Se0 and entrapment of Bio-Se0 by different size groups of aerobic granules were investigated to optimize biological treatment process for Se-laden wastewaters. Furthermore, a bacterial strain showing high selenite tolerance and reduction was isolated and characterized. Removal of selenite and conversion to Bio-Se0 were achieved by all the size groups of granules ranging from 0.12 mm to 2 mm and above. However, selenite reduction and Bio-Se0 formation were rapid and more efficient with large aerobic granules (≥0.5 mm). The formed Bio-Se0 was majorly associated with the large granules, due to better entrapment capabilities. In contrast, the Bio-Se0 formed by the small granules (≤0.2 mm) was distributed both in the granules and aqueous phase because of ineffective entrapment. Scanning electron microscope and energy dispersive X-ray (SEM-EDX) analysis confirmed formation of Se0 spheres and association with the granules. Efficient selenite reduction and entrapment of Bio-Se0 was related to prevalent anoxic/anaerobic zones in the large granules. A bacterial strain showing efficient SeO32- reduction of up to 15 mM SeO32- under aerobic conditions was identified as Microbacterium azadirachtae. SEM-EDX analysis confirmed the formation and entrapment of Se0 nanospheres (size: 100 ± 5 nm) in the extracellular matrix. The cells immobilized in alginate beads showed effective SeO32- reduction and Bio-Se0 entrapment. Efficient reduction and immobilization of bio-transformed metalloids by large AGS and AGS-borne bacteria implicates prospective use in bioremediation of metal(loid) oxyanions and bio-recovery.

Effects of oxytetracycline on aerobic granular sludge process: Granulation, biological nutrient removal and microbial community structure.

Formation of aerobic granular sludge (AGS), process performance and microbial community structure were investigated in lab-scale sequencing batch reactors (SBR) operated without and with oxytetracycline (OTC). Granulation of activated sludge and appearance of AGS was observed in parallel SBRs operated without and with OTC. However, formation of well-settling aerobic granules was relatively faster in the SBR fed with 100 µg/L OTC and observed within 2 weeks of start-up. Ammonium, total nitrogen, and phosphorus removals were quickly established in the AGS cultivated without OTC. In contrast, nitrogen and phosphorus removals were lower in the OTC fed SBR. But, a gradual improvement in nitrogen and phosphorus removals was observed. After 45 days, nitrogen and phosphorous removals were stabilized at 99% and 70%, respectively, due to establishment of OTC-tolerant community. qPCR revealed the impact of OTC on ammonium oxidizing bacteria, polyphosphate accumulating organisms and their enrichment during exposure to OTC. Ammonium and phosphorus were majorly removed via nitritation-denitritation and enhanced biological phosphorus removal (EBPR) pathways, respectively, in the presence of OTC. Brevundimonas (35%), Thaurea (14%) sp. Ca. Competibacter (5.6%), and Ca. Accumulibacter (4.2%) were enriched in OTC-fed AGS. Of the two OTC-tolerant strains isolated, Micrococcus luteus exhibited growth and efficient OTC biotransformation at different OTC concentrations. Moreover, M. luteus was predominantly growing in the form of aggregates. Key traits such as tolerance, biotransformation and high autoaggregation ability allowed a niche for this strain in the granules. This work has important implications in understanding the effect of antibiotics on AGS and designing AGS based treatment for antibiotic-laden wastewaters.

Enhanced biological phosphorus removal in aerobic granular sludge reactors by granular activated carbon dosing.

This study investigated the effects of granular activated carbon (GAC) addition on the enrichment of polyphosphate accumulating organisms (PAOs), stratification of PAOs in the co-existing GAC-biofilms and granules and biological nutrient removal (BNR) in aerobic granular sludge (AGS) reactors. It was found that BNR increased in the GAC-augmented system. Establishment of enhanced biological phosphorus removal (EBPR) pathway was faster with about 1.7 to 2-fold higher P removal in GAC system than control. EBPR biomass grown in the presence of GAC was segregated into different size fractions for determining BNR and stratification of microbial groups. It was found that EBPR was majorly associated with the large biomass (>0.5 mm) fraction, corroborating with higher abundance of PAOs. Higher P removals of 60 to 70% with characteristic EBPR profiles were observed in 0.5 mm fraction. In contrast, P removals by 0.25 mm fraction were lower at 20 to 35% without EBPR profiles. EBPR biomass (>0.5 mm) fraction was segregated into granules and GAC-biofilms for determining the role of GAC in PAOs enrichment. P release (2.5-3.5 mg L-1 P) and P uptake (5-7 mg L-1 P) were higher in the P removal profiles exhibited by GAC-biofilms. In contrast, P release and P uptake were lower with the granules. These differences in P removal profiles resulted in distinct net P removal efficiencies of 70 ± 5% and 50 ± 6% for GAC-biofilms and granules, respectively. These differences in P removals were corroborated by higher abundance of PAOs in the GAC-biofilms than co-existing granules. PAO clade-level enrichment was found to be dependent on substrate wherein acetate feeding enriched PAO clade I, while acetate-propionate feeding caused enrichment of both PAO clade I and II. These results suggest that GAC addition to AGS reactors can aid in enrichment of PAOs, reduce the start-up period for EBPR, and increase P removal efficiencies.

Aerobic granular sludge for efficient biotransformation of chalcogen SeIV and TeIV oxyanions: Biological nutrient removal and biogenesis of Se0 and Te0 nanostructures.

Simultaneous removal of selenite (SeIV), tellurite (TeIV) and nutrients by aerobic granular sludge (AGS) was investigated. A sequencing batch reactor (SBR) was operated with increasing SeIV and TeIV (up to 500 µM each) for 205 days to evaluate metalloid oxyanion and nutrient removal. AGS efficiently removed SeIV and TeIV by readily converting them to biomass associated forms. The total Se and Te removal efficiencies were higher at 98% and 99%, respectively. Formation of biomass-associated Se0 and Te0 was confirmed by XRD, Raman spectroscopy and SEM-EDX. Feeding of SeIV and TeIV elicited inhibitory action on ammonium removal initially, nonetheless removal performance was recovered during the subsequent cycles. Ammonium, total nitrogen and phosphorus removals were stabilized at 85%, 80% and 75%, respectively, at 500 µM of SeIV and TeIV. Sequencing of 16S rRNA gene confirmed enrichment of known SeIV and TeIV reducing bacteria in the granules. qPCR and removal kinetics supported ammonia removal via nitritation-denitritation. This work demonstrates functional capabilities of AGS for effectively removing toxic SeIV and TeIV oxyanions apart from performing simultaneous COD, nitrogen and phosphorus removal. Efficient biological nutrient removal in the presence of toxic SeIV and TeIV concentrations, suggests robustness of AGS and its resilience to toxic contaminants.

Enhancing biological nitrogen and phosphorus removal performance in aerobic granular sludge sequencing batch reactors by activated carbon particles.

Long start-up periods for aerobic granular sludge (AGS) formation and establishment of P removal pathways are challenges for widespread implementation of AGS process. External additives such as activated carbon (AC) attracted interest for accelerating AGS formation. However, the roles of AC in granulation and biological nutrient removal (BNR) are not understood. Here, the role of AC was investigated in decreasing start-up periods in AGS formation and BNR under different carbon substrate conditions (i.e., acetate (HAc), propionate (HPr) and HAc-HPr) in sequencing batch reactors (SBRs). AC addition increased aggregation index and settleability of activated sludge (AS) inoculum which minimized AS washout from SBRs. AC addition hastened AGS formation and establishment of BNR pathways by facilitating AS retention and biofilm formation. Feeding HAc or HAc-HPr supported better granulation (MLSS: 6-7 g l-1, SVI: 30-40 ml g-1) than HPr (MLSS: 4 g l-1, SVI: 70). The start-up periods for efficient total nitrogen (TN) removals were decreased to 22 and 16 d from 38 to 25 d, respectively, in AC augmented SBRs fed with either HAc or HAc-HPr. TN removals were higher at ≥95% in HAc or HAc-HPr fed SBRs. Total phosphorus (TP) removals were also higher in AC-augmented SBRs at 80% and ≥90% in HAc and HAc-HPr fed SBRs, respectively. In contrast, TN and TP removals were lower at 70% and 35%, respectively, in HPr fed SBR. Ammonium was primarily removed via nitritation-denitritation pathway. Phosphorus removal was at 1.7 to 2-fold higher in AC augmented SBRs and driven by enhanced biological phosphorus removal (EBPR) pathway. MiSeq sequencing and qPCR revealed higher enrichment of polyphosphate accumulating organisms (PAOs), denitrifying PAOs, and ammonia oxidizers in AC-augmented SBRs fed with HAc or HAc-HPr. This study demonstrates that AC addition can be considered for enrichment of PAOs and establishment of EBPR in aerobic granular SBRs.

Biological nutrient removal by halophilic aerobic granular sludge under hypersaline seawater conditions.

Biological nutrient removal and physical properties of halophilic aerobic granular sludge (hAGS) cultivated from autochthonous seawater-born microbes were investigated under hypersaline seawater conditions. hAGS achieved stable total nitrogen (TN) and total phosphorus (TP) removals of 96 ± 3% and 95 ± 4%, respectively, from seawater-based wastewater at 3.4% salt. At 4 to 12% salt concentrations, stable TN and TP removals of 82-99% and 95-96%, respectively, were maintained over 4 months under seawater conditions. Ammonium and phosphorus were mainly removed by nitritation-denitritation and enhanced biological phosphorus removal pathways, respectively. Stappiaceae (45%) and Rhodobacteraceae (21%) were the dominant genera in hAGS performing nutrient removal at 12% salt. hAGS contained acid-soluble extracellular polymeric substance as the major structural polymer which increased from 0.43 ± 0.02 g/gTS at 3.4% salt to 0.93 ± 0.03 g/gTS at 12% salt. Cultivation of hAGS from autochthonous wastewater-microbes can be a promising approach for achieving biological nitrogen and phosphorus removals from hypersaline seawater-based wastewaters.

Aerobic granular sludge for high-strength ammonium wastewater treatment: Effect of COD/N ratios, long-term stability and nitrogen removal pathways.

Aerobic granular sludge (AGS) technology is increasingly considered for wastewater treatment. AGS stability particularly under lower COD/N ratios is an impediment for AGS technology. This study evaluated AGS stability and nitrogen removal at different loading rates of 0.03 to 4 kg NH4+-N m-3 d-1 and COD/N ratios of 18.3 to 0.13. Ammoniacal and total nitrogen removals were high at 99.9% and 99.3%, respectively, during 440 days. MiSeq sequencing revealed a reduction in bacterial diversity and enrichment of ammonia oxidizing bacteria (AOB), anammox and denitrifying bacteria. Quantitative PCR showed enrichment of AOB, anammox bacteria, Nitrospira and denitrifiers. Chemical data and bacterial community supported occurrence of nitritation and anammox pathways. AGS had stable granular structure with excellent settling properties at lower COD/N ≤ 1. Removal of high-strength ammonium could be partly explained by the existing nitrogen pathways suggesting novel mechanisms. Nevertheless, results presented here support implementation of AGS process for ammonium wastewaters.

Textile dye biodecolourization and ammonium removal over nitrite in aerobic granular sludge sequencing batch reactors.

Biodecolourization of azo dye and removal of ammonium by aerobic granular sludge (AGS) was investigated under different growth conditions. AGS not previously exposed to azo dye was able to effectively decolourize azo dye under anaerobic and microaerophilic conditions. Azo dye, total organic carbon and ammoniacal nitrogen removal efficiencies of 89-100%, 79-95% and 92-100%, respectively, were achieved in the AGS reactor operated for 80days under microaerophilic conditions. Removal of carbon, nitrogen and phosphorus was not impacted by azo dye loading. Azo dye, organic carbon and ammonium were majorly removed in the anoxic period wherein bulk dissolved oxygen was ranged from 0.5 and <0.08mgL-1. Removal of 60mgL-1 NH4+-N was associated only with smaller amounts of nitrite build-up (∼5mgL-1 NO2--N) and negligible nitrate concentrations. Profiles of nitrogen compounds in individual sequencing batch reactor cycles supported the occurrence of ammonium removal over nitrite pathway. Bacterial community analysis showed enrichment of specific microorganisms capable of decolourizing azo dyes in the dye-decolourizing AGS. Dye decolourization and nutrient removal by AGS under microaerophilic conditions is a novel finding and can be further developed for treating textile wastewaters onsite or after dilution with sewage.

Selenite reduction and ammoniacal nitrogen removal in an aerobic granular sludge sequencing batch reactor.

Simultaneous removal of selenite and ammonium by aerobic granular sludge was investigated to develop an improved biological treatment process for selenium rich wastewaters. Aerobic granules not previously exposed to selenite were able to remove selenite by converting it to elemental selenium (Se(0)) and simultaneously remove ammonium under different conditions in batch experiments. To achieve sustainable selenite and ammonium removal, an aerobic granular sludge reactor was operated in fill-and-draw mode with a cycle of anaerobic (8 h) and aeration (15 h) phases. Almost complete removal of different initial concentrations of selenite up to 100 µM was achieved in the anaerobic phase. Ammonium removal was severely inhibited when the granules were initially exposed to 1.27 mg L-1 selenite, but ammonium and total nitrogen removal efficiencies gradually improved to 100 and 98%, respectively, under selenite-reducing conditions. Selenite loading shifted ammonium removal occurring mainly during the anaerobic phase to both the anaerobic and aeration phases. Selenite was removed from the aqueous phase by converting it to nanoparticulate Se(0), which was entrapped in the granular sludge. Scanning electron microscop-energy dispersive X-ray spectroscopy and X-ray diffraction analysis confirmed the formation of Se(0) nanospheres and their retention in the granular sludge. The effluent Se ranged from 0.02 to 0.25 mg Se L-1, while treating up to 12.7 mg L-1 selenite, which is lower as compared to previous studies on selenite removal using activated sludge or anaerobic granular sludge. This study shows that aerobic granular sludge reactors are not only capable of removing toxic selenite, but offer improved treatment of Se-rich wastewaters.