Eco-friendly Upcycling of Spent Coffee Ground and Treatment of Recalcitrant Wastewater Using Microbial Consortia

Abstract

Spent coffee grounds (SCG), one of the world's most discarded wastes, could be an excellent resource to make a good organic fertilizer because of its richness in organic nutrients. SCG, poultry manure, and agricultural waste-derived biochar were used to manufacture functional composts through microbial bioaugmentation. The research objective is to employ fermentation utilizing biochar and beneficial microorganisms and manufacturing a functional compost product of an eco-friendliness and high quality. The highest yield of tomato stalk- based biochar (40.7%) was obtained at 450°C with a surface area of 2.35 m2/g. Four pilot scale composting reactors were established to perform composting for 45 days. The ratios of NH4+-N/NO3--N, as an indicator of compost maturity, indicate a rapid and successful composting via microbial bioaugmentation and the biochar amendment. Moreover, germination indices for radish also increased by 14 -34% through the augmentation and the biochar amendment. The microbial diversity was also enhanced in the augmented and biochar-amended composts by 7.1-8.9%, where two species of Sphingobacteriaceae were dominant (29-43%). DPPH scavenging activities were enhanced by 14.1% and 8.6% in fruits of pepper plants grown in presence of the compost TR-2 (augmentation only) and TR-3 (biochar amendment plus augmentation), respectively. Total phenolic content was also enhanced by 68% in fruits of the crop grown in TR-3. Moreover, the other compost TR-L (augmentation only) boosted DPPH scavenging activity by 111% in leek compared with the commercial organic fertilizer, while TR-3 increased the phenolic content by 44.8%. The composting facilitated by microbial augmentation and biochar amendment could shorten the composting time and enhance quality of the functional compost. These results indicate that the functional compost has a great potential to compete with the commercially available organic fertilizers and the novel composting technology could significantly contribute to eco-friendly recycling of organic wastes such as spent coffee grounds, poultry manure and agricultural wastes. The next topic of the thesis was to investigate a synergistic relationship between carbon degradation and denitrification pathways in the full-scale tannery wastewater treatment plant bio-augmented with a microbial consortium BM-S-1. It was hypothesized that denitrification process closely related to the degradation of amino acids and fatty acids which could generate electron donors for the nitrate reduction, and that an efficient removal of nitrogen was linked to COD removal (and hence sludge reduction). The goal of this study was to elucidate the relationships between denitrification and degradation pathways for amino acids and fatty acids through functional metagenomic analysis. Shotgun metagenomic reads were mapped to 'ChocoPhlAn' pan-genome database and MetaPhlAn2 database for organism-specific functional profiling and formed as a gene abundance data using HUMAnN2. We then compared and selected the gene families and pathways using the extended databases UniProt Reference Clusters (UniRef90, http:/www.uniprot.org) and MetaCyc metabolic pathway database. During the functional gene analysis, 40,181 gene families and 196 pathways were revealed for the five treatment stages B, PA, SA, SD, and I. The metagenomics analysis of the microbial community showed that Brachymonas denitrificans, a known denitrifier, highly occurred in B, PA and SD. The occurrences of the amino acid degrading enzymes, alpha ketoglutarate dehydrogenase (α- KGDH) and tryptophan synthase, highly correlated with those of the denitrification genes such as napA, narG, nosZ and norB. The occurrence of glutamate dehydrogenase (GDH) also highly paralleled with those of the denitrification genes such as napA, narG, and norZ. The denitrification genes (nosZ, narG, napA, norB and nrfA) and amino acid degradation enzymes (tryptophan synthase, α- KGDH and pyridoxal phosphate dependent enzyme) were dominantly observed in B. This indicates that degradation of the substrates (proteins) and denitrification of ammonium may occur actively in the treatment stage. The high abundance of fatty acid degradation enzyme groups (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and beta-ketotiolase) was observed together with the denitrification genes like napA, narG and nosZ. Like amino acid degradation enzymes, more diverse enzymes were dominantly observed in the stage B such as phospholipase/carboxylesterase, enoyl-CoA hydratase/isomerase, acyl-CoA dehydrogenase, phenylacetate degradation, and 3-hydroxyacyl-CoA dehydrogenase 2. All these results clearly state that the denitrification pathways could be linked with degradations of the amino acids and fatty acids whose degradation products go through TCA cycle to generate NADH that is used as electron donors for the denitrification. This understanding of nitrogen and carbon metabolic pathways in the tannery wastewater treatment system will undoubtedly contribute to an optimized and efficient operation of the treatment system and any other wastewater treatment systems. The last topic of the thesis was to evaluate the degradation activities of pollutants in the dye wastewater treatment process in terms of treatment efficiency and to provide metagenomic understanding of the treatment process. Hypothesis is that the azo-dye could be degraded via pathways including azo-dye reduction, aromatic compound degradation, nitrification, denitrification, and TCA cycle. The activities of dye wastewater treatment plant were evaluated before and after the addition of CES-1. Total genomic DNA samples were extracted from different treatments with different time variables. The effect of CES-1 bioaugmentation on the microbial community structure and functional genes of each treatment step was explored by high-throughput sequencing and metagenomic analysis. The removal efficiencies (%) for COD, T-N, T-P, SS and color intensity before bioaugmentation were 94.9, 48.1, 91.6 63.9 and 66.3, respectively. By the way, the removal efficiencies (%) for these parameters 50 days after the bioaugmentation increased up to 97.8, 62.6, 95.8, 63.4 and 77.9, respectively. The sludge reduction rate over 20 months after the bioaugmentation was 26% (reduction from 6.18 to 5.00 in sludge per ton of influent COD). There appeared to be no clear delineation in the microbial communities between processes of influent (I) (I_0310_18) and buffering (B) (B_0310_18). The buffering sample (B_0629_17) maintained quite a different community structure from the influent sample (I_0629_17), indicating the modifying effect of CES-1 bioaugmentation on the buffering process despite the very high domination of Mesorhizobium soli (98.5%) in the influent. The presence of FMN dependent NADH-azoreductase were dominant before treatment (B_0303_17), but a drastic decrease of the enzyme was observed in B after CES-1 treatment, indicating that a competition occurs between microbial communities from CES-1 and the indigenous organisms carrying azoreductase. Enzymes involved in aromatics degradation were generally present in the samples (B_0310_18, PA_0310_18, SA_0310_18 and SD_0310_18) 300 days after bioaugmentation while frequencies of these enzymes were quite low before and 50 days after augmentation, indicating the selection of these aromatic degradative enzymes by CES-1 augmentation over time. CES-1 augmentation also delineated samples into the two groups: the samples of control and early stage of bioaugmentation (B_0303_17 and I_0629_17) and the samples of later stage of bioaugmentation (PA_0310_18). Dominant amino acid degradative enzymes found in the later stage of the augmentation were 2-hydroxyglutarate dehydrogenase, tryptophan synthase, 2-oxoglutarate dehydrogenase and acetylglutamate kinase. The representative enzymes for TCA cycle found in later stage of the augmentation were succinate dehydrogenase (ubiquinone) iron-sulfur subunit, succinate dehydrogenase, triosephosphate isomerase and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase. Enzymes involved in nitrogen metabolism dominantly found in the later stage of the augmentation were nitrite reductase, hydroxylamine dehydrogenase, nitrous oxide reductase, nitrate reductase/nitrite oxidoreductase, nitrite reductase, periplasmic nitrate reductase, ammonia monooxygenase subunit A and nitrite reductase. The intrinsic relationships between microbial community structures and functions of enzymes for the dye metabolism need to be analyzed using more sophisticated algorithms. Since the successful performance in reduction of pollution and sludge in the dye wastewater treatment plant and the concomitant metagenomic analysis of the treatment process was accomplished, the future perspective of this research will be the development of specific genes and enzymes as biomarkers used to monitor the functional properties of degradation and operate the novel dye wastewater treatment system in the optimal conditions.