3.1 Physico-chemical Analysis of Leachate-
Physico-chemical characterization of leachate samples showed an extremely low BOD/COD ratio (0.17), which is a typical characteristic of mature/ sanitary leachate and also defines its low biodegradable nature (Table- S1) (Wu et al. 2020; Wang et al. 2021) with slightly alkaline pH-7.5 (Yu et al. 2014; Ardeshir et al. 2017). This also signifies that leachate contains complex inorganic chemicals, which might be recalcitrant in nature. These can be highly toxic for inhabitant microbiota as well as prevalent environment. pH (>7.5) also indicates the maturation stage of the leachate. Elevated EC content (17.58-36 ms cm-1) indicates high salinity of leachate sample. TDS and nitrogen concentration was also found very high. Environmental release of toxic leachate will eventually disturb the hydro-geological balance, existing microflora, and enzymatic properties of soil and hence will affect its productivity. Figure- 3b shows the overall distribution pattern of different pollutants in raw and primary treated leachate (PT), in raw leachate 89% of the pollutant load is shared among COD (42%) and TDS (47%) rest of the parameters share <10% of the pollutant load. Whereas, in the primary treated sample TDS load is decreased to 22%, hence COD possessed the maximum pollutant load of 59%.
3.2 Isolation & Screening of Indigenous Microorganisms
Along with toxicity and potential environmental threat, low BOD/COD ratio of primary treated leachate also signifies dearth of microflora. But it can be assumed that the surviving microbial isolates would be adaptive to the surrounding toxins and hence are supposed to metabolize them for their growth. Therefore the present study emphasizes on identification and characterization of indigenous robust microbes from the complex leachate matrix.
3.2.1 Survival Competency
Raw and primary treated leachate respectively contained 45*106 and 25*106 CFU of initial bacterial load which reduces by > 90% on the first cycle of enrichment and further increased up to 99.99% after four consecutive cycles. Finally, 16 distinct bacterial colonies (11 from raw + 5 from primary treated leachate) and 2 fungi were isolated and characterized for the further metabolic screening process. The drastic decrease in the microbial population can be attributed to the presence of toxic recalcitrant chemicals in the sanitary leachate. The morphological characterization of bacteria shows that most of them belong to the genus Bacillus, whereas for fungi, one was sporulating (F1) while the other was having mycelia (M1). In concurrence to our findings Remmas et al. 2017 also found Bacillales as a minor but important constituent of indigenous leachate microflora identified by culture-dependent Illumina sequencing. They explored that >42% microflora of a mature leachate belonged to Candidimonas, Pusillimonas, Leucobacter, Paralcaligens, Castellaniella, Eoetvosia, Parapusillimonas, and Pseudomonas. In contrast, Gammaproteobacteria, Firmicutes, and Bacteroidetes dominated in middle-aged or young landfill leachate (Song et al. 2015b). Williams and Hakam, 2016; Siddiqqui, 2017; Morris et al. 2018 had also isolated various bacterial species from landfill sites.
Resilient microbes make landfill leachate an unexplored source for novel biotechnological applications. They can be used for designing strategical wastewater treatment which results in significant reduction of recalcitrant pollutant load. Similarly Rennas et al. 2017 have also characterized some extremophiles; halotolerant alkaliphiles belonging to the genera Dietzia, Glycocaulis, Halomonas, Marinobacter, Piscibacillus, and Rhodobacter from mature landfill leachate.
For fungal cultures, 10 distinct fungi were identified in both leachate samples (raw and primary treated) which reduced up to 80% after four cycles of enrichment culture; two distinct surviving fungi were morphologically characterized. Bacterial cultures were termed as C1- C16 (C1, C2, C3,…….., C16), and fungal cultures were termed as F1 & M1. Detailed molecular characterization was done after screening efficient strains amongst them.
3.2.2 Metabolic Competency
Leachate used in the present study shows high COD and low BOD values and hence carbon degradation efficiency has been used as a tool for the screening of potential isolates (Saraswathy et al. 2001; Saidi et al. 2011). This implies their capability to break the complex chain of hazardous toxic chemicals into simpler compounds upon which they can feed later on. The graph shows that bacterial isolates C2, C3 & C4 showed highest TOC degradation potential of 38.27, 44 & 37.6 % respectively within 2 days of incubation (Figure S2). These were followed by C7, C5, and C8, which shows degradation rates of 31, 28 & 23.5 % respectively. Control samples showed negligible rate of degradation. Hence the first three bacterial isolates, C2, C3 & C4, were selected for further characterization. Both fungal isolates did not show considerable TOC degrading efficiency and hence were not considered further.
In contrast to our study Saidi et al. 2011 has screened Bacillus, Actinomyces, Pseudomonas, and Burkholderia genus from a year-old landfill site in Tunisia with 60-90% to carbon degrading efficiency within 48 hours. This may be due to the difference in leachate composition as the tested leachate sample was from a young landfill with abundant carbon content and hence could be easily metabolized by native microbes. But in the present study, BOD/COD ratio is quite less and hence the microbes can only survive by breaking the complex bonds, which can be time-consuming, hence the rate of carbon reduction was found significantly low. Primary morphological characterization revealed that all the three isolates belong to most predominant resilient bacterial genera, Gram-negative cocci.
3.3 Molecular Characterisation of Screened Isolates
Screened isolates were subjected to 16S rDNA sequencing. The obtained sequences were used for the construction of phylogenetic tree for identification (Figure S3; Table S2). The screened isolates find their closest similarity to Leclercia spp., Pseudescherichia spp. and Brucella spp. for strains C2, C3, and C4 respectively. The sequences were submitted to NCBI Gene Bank with Accession numbers MT186162, MT186163 & MT186164. To our knowledge, this is the first study that reports the presence of all these three bacterial strains in sanitary leachate. Bacillus and Pseudomonas spp. are known to be common inhabitants of contaminated sites and have been previously studied for their aromatic hydrocarbon degradation potential by Hale et al. 2001.
In concurrence with our study, Huang et al. 2005 have also explored the microflora of an MSW leachate by using molecular tools and found the presence of 76 sequence types representing 138 randomly selected non-chimeric clones. Siddique, 2017 has also studied molecular characterization of the microflora of landfill leachate and observed the presence of different genera Halomonas, Lueitomonas, Bacillus, and Bordetella, etc.
3.4 Metabolic potential of Leachate isolates
Metabolically efficient strains can be used for evaluating their pollutant reduction potency under in-situ conditions. The metabolic potential of the isolated strains was comparatively slow in the inoculated leachate matrix; 60-80% of the pollutant load was reduced within the treatment time. A booster dose of active indigenous microbial culture increases their metabolic potential resulting in >90% reduction within the stipulated time frame (Figure- 2a). Relative residual pollutant concentration also correlates with our initial findings, which was significantly high in inoculated samples as compared to augmented ones. Major impact was noticed for BOD and COD as depicted in figure- 2b. Hence we can say that pre-incubated microbes or their consortia work as a catalyst in increasing the metabolic growth rate of existing microflora. Amakdouf et al. 2022 had isolated and screened eight indigenous strains based on their heavy metal tolerance profile, which reduced the pollutant load significantly by augmentation. Under optimum conditions, the COD removal rate increased to 86.0% and 90.0%, after treatment with E. casseliflavus Jlu and B. cereus Jlu after 2 days of degradation (Yu et al. 2014).
Hence in the present study, the three characterized strains were augmented into the leachate matrix and their pollutant reduction potential was studied in a timeline pattern & represented in the form of the Leachate Pollution Index (LPI). Bio-augmentation has already been reported as a suitable pre-treatment technology for increasing the biodegradability of landfills (Mishamandani et al. 2016; Styriakova et al. 2016; Kadri et al. 2017).
3.4.1 Timeline view Residual Pollutant concentration
Residual pollutant concentration at different point of times 0, 7, 21 and 41 days after bioaugmentation treatment by indigenous strains is shown in figure 3a.
pH of the medium showed a bell shaped curve i.e. increases upto a certain period to alkaline side and afterwards ended into an acidic liquid. The initial pH of the medium was found to be 7.0, which increased initially up to 21 days in the range of 9-11.5 by all the strains and then decreased to 8.8-9.1 during 21-41 days. This depicts the characteristic biodegradation pattern in which there is a strategic degradation of contaminants into some metabolites and by-products (which may be alkaline in nature) but ultimately yields to the production of carbon dioxide and acids, which led to a sharp decrease in the pH of the sample.
Pollutant parameters viz. BOD, TDS, and phenols degrade at a slower rate in the initial days and then a steep decrease was observed. This can be explained in the purview of microbial growth, lag phase. The lag phase is actually the adaptation period for the augmented culture, during which the microbes acclimatize to the prevalent environment, and also incompetent microbes may get screened. Once they are fully adapted to the matrix, they show their maximum metabolic potential which is reflected in the form of steep increase in the percent reduction.
At the end of the experiment residual concentration was found to be 20-23, 140-155, 74, 0.8, 400-470, 315-430 mg L-1 for BOD, COD, TKN, Phenols, TDS, and chlorides respectively.
Microbial reduction significantly varied (p<0.05) among all the three isolates in the order BC3 (F(p<0.05)= 255.4)> BC2 (F(p<0.05)=131.4) >BC4 (F(p<0.05)=26.4). Variation in degradation pattern can be explained in view of the nutritional preference of bacteria for survival. Also, it can be explained in view of toxic contaminants or their first stage metabolic by-products, which may inhibit microbial activity in due course of time (Jain et al. 2013). A detailed molecular analysis will further be required to find out the metabolic pathways of individual contaminants present in the leachate.
Individual percent reduction for pollutant parameters was found to be in the range of 89-90, 87-88, 86-87, 88-89, 64-78, 15-26% respectively for BOD, COD, TKN, Phenols, TDS, and chlorides. In concurrence Heang et al. 2020 had reported variable degradation efficiency of Alcaligenes faecalis with respect to different BOD/COD ratios. They also deferred that the rate of degradation reduced drastically by 50-80% at a low BOD/COD ratio of 0.5-0.7. Most of leachate treatment studies have been done in high organics containing leachate which is totally different from our matrix hence the treatment rate may vary (Fatma et al. 2016; Zegzouti et al. 2020; Saetang and Babel 2012). Apart from these, various scientists have used microbial treatment in combination with other physicochemical methods for the treatment of landfill leachate (Galvez et al. 2009; Feng et al. 2019; Heang et al. 2020; Yasmin et al. 2020; Song et al. 2020; Rani et al. 2020) and observed reduction in major pollutant variables. Experimental studies on microbial treatment of low organics containing leachate with overall quality analysis are very few (Zegzouti et al. 2020).
As per the overall pollutant distribution pattern, 59% of the initial COD load was reduced up to 17, 14, and 19% respectively by BC2, BC3, and BC4. Major pollutant load (>66%) was shared among chlorides and TDS by all the three isolates after treatment (Figure- 3b). Rest parameters share 15-17% of the pollutant load after augmentation treatment. Microbial growth well correlated with the reduction potential in both inoculated and bio-augmented leachate matrix as seen in figure- 4c, this states that microbes intake residual pollutants for their growth and metabolism hence exhibits high growth, and higher metabolic potential. In our previous study also we had found a positive correlation between the growth and pesticide degradation potential of indigenous fungi in a liquid medium (Jain et al. 2012).
3.4.2 Leachate Pollution Index (LPI)
LPI is a new interactive tool to study the cumulative pollution matrix of a particular site (Jain et al. 2021; Rafijul and Alamgir, 2013; Naveen et al. 2016). The LPI value of primary treated leachate was found to be 13.39, which decreased by 28-42% by inoculated strain treatment (C2, C3, C4) whereas in the bio-augmented sample (BC2, BC3, BC4) the decrease was exponential up to 61% (approx.) after 41 days of treatment (table-S3). LPI value in the individual treated sample was relatively higher than the standard discharge limit as depicted in figure- 4, but bio-augmented strains showed higher metabolic potential by decreasing the LPI to 5.1-5.2 which is significantly lower than the standards (7.4).
Bhalla and Jha, 2014 had treated the landfill leachate by passing through a permeable reactive barrier and reduced the LPI value from 26.45 to 7.03, which is acceptable for environmental discharge MOEF, 2000.
3.5 Degradation Kinetics
Microbial degradation kinetics is a measure to envisage their metabolic potential towards pollutant reduction, its correlation with pollutant concentration, and ultimately its residence time in the matrix. Detailed kinetic graphs are also shown in Figure- S4. Based on the optimum R2 value the observed data was best fitted into the first-order equation for all the six tested parameters; BOD, COD, chlorides, TDS, TKN, and phenol as well as three augmented strains. Hence we can say that microbial metabolic potential is directly correlated with that of pollutant concentration. In concurrence with our study, Ahmadian et al. 2013 had studied Fenton process for the treatment of municipal landfill leachate and observed best fit model technique to find a suitable match.
Kinetic constants were found to be in the order of BC4< BC3< BC2, for microbial metabolic efficiency & chlorides< phenols< TDS< TKN< BOD< COD for tested pollutant parameters. The half-life for all the tested parameters will be exactly the reverse of this. Linear regression plots for microbial reduction were found well fitted within the first-order equation; hence we can say that observed values are quite comparable with that of model values. Kinetic coefficients of microbial degradation are depicted in table- 1.
Sekman et al. 2011 had studied first-order degradation kinetics of phenols in landfill leachate under anaerobic conditions. In our previous research, we had also observed first-order degradation kinetics for fungal degradation of an organophosphorus compound, Monocrotophos (Jain et al. 2013).
Regression coefficient (R2), RMSE and SSE values were observed to correlate the predicted values with that of obtained data. RMSE value was found <0.5 for BOD, COD, Chloride, TDS, and TKN whereas for phenols it was found slightly high 0.62-0.65. This also deciphers that the applied model exhibits close similarity with that of the standard response. R2 value was also found significant at p<0.01.
Table- 1 Kinetic Coefficients for Microbial degradation kinetics
Pollutant Parameters |
BC2 |
BC3 |
BC4 |
k |
t1/2 |
R2 |
SSE |
RSME |
k |
t1/2 |
R2 |
SSE |
RSME |
k |
t1/2 |
R2 |
SSE |
RSME |
BOD |
0.073 |
9.49 |
0.96*** |
0.41 |
0.32 |
0.053 |
13.08 |
0.99*** |
0.03 |
0.08 |
0.057 |
12.16 |
0.99*** |
0.01 |
0.04 |
COD |
0.081 |
8.56 |
0.98*** |
0.12 |
0.17 |
0.082 |
8.45 |
0.98*** |
0.13 |
0.18 |
0.079 |
8.77 |
0.99*** |
0.06 |
0.11 |
Chloride |
0.008 |
86.63 |
0.99*** |
0.00 |
0.01 |
0.008 |
86.63 |
0.99*** |
0.00 |
0.01 |
0.010 |
69.30 |
0.99*** |
0.00 |
0.15 |
TDS |
0.031 |
22.35 |
0.99*** |
0.00 |
0.02 |
0.036 |
19.25 |
0.99*** |
0.01 |
0.06 |
0.025 |
27.72 |
0.99*** |
0.01 |
0.04 |
TKN |
0.048 |
14.44 |
0.99*** |
0.01 |
0.06 |
0.049 |
14.14 |
0.99*** |
0.00 |
0.03 |
0.050 |
13.86 |
0.99*** |
0.02 |
0.07 |
Phenols |
0.024 |
28.88 |
0.89*** |
1.69 |
0.65 |
0.025 |
27.72 |
0.90*** |
1.55 |
0.62 |
0.024 |
28.88 |
0.89*** |
1.52 |
0.62 |
***significant at p<0.01; SSE- Sum of Standard Error; RSME- Root Square Mean Error
3.6 AMES test
Secondary treated leachate samples showed no mutagenicity, hence can be considered environmentally safe for discharge.
Table 2 Mutagenicity of Secondary treated Leachate samples
Sample |
TA98 |
TA 100 |
BC 2 |
23 |
95 |
BC 3 |
15 |
96 |
BC 4 |
27 |
103 |
Negative control |
28 |
120 |
Positive control |
76 |
258 |
3.7. Correlation Analysis
Cumulative pollution ratings were correlated for each individual parameter tested and among the different samples (before and at each treatment step, the obtained results show that all the analyzed parameters were found to be well correlated (Table S4). Total phenols showed a strong correlation factor after microbial treatment. Only TKN and TDS in primary treated samples showed nil correlation with an r=0.
In intra-treatment statistics, BOD showed an almost perfect correlation with all strains.COD also showed a good correlation, none of the parameters were found uncorrelated/non-correlated (Table S5).