Regulating microbial redox reactions towards enhanced removal of refractory organic nitrogen from wastewater.

Biotechnology for wastewater treatment is mainstream and effective depending upon microbial redox reactions to eliminate diverse contaminants and ensure aquatic ecological health. However, refractory organic nitrogen compounds (RONCs, e.g., nitro-, azo-, amide-, and N-heterocyclic compounds) with complex structures and high toxicity inhibit microbial metabolic activity and limit the transformation of organic nitrogen to inorganic nitrogen. This will eventually result in non-compliance with nitrogen discharge standards. Numerous efforts suggested that applying exogenous electron donors or acceptors, such as solid electrodes (electrostimulation) and limited oxygen (micro-aeration), could potentially regulate microbial redox reactions and catabolic pathways, and facilitate the biotransformation of RONCs. This review provides comprehensive insights into the microbial regulation mechanisms and applications of electrostimulation and micro-aeration strategies to accelerate the biotransformation of RONCs to organic amine (amination) and inorganic ammonia (ammonification), respectively. Furthermore, a promising approach involving in-situ hybrid anaerobic biological units, coupled with electrostimulation and micro-aeration, is proposed towards engineering applications. Finally, employing cutting-edge methods including multi-omics analysis, data science driven machine learning, technology-economic analysis, and life-cycle assessment would contribute to optimizing the process design and engineering implementation. This review offers a fundamental understanding and inspiration for novel research in the enhanced biotechnology towards RONCs elimination.

Occurrence, transmission and risks assessment of pathogens in aquatic environments accessible to humans.

Pathogens are ubiquitously detected in various natural and engineered water systems, posing potential threats to public health. However, it remains unclear which human-accessible waters are hotspots for pathogens, how pathogens transmit to these waters, and what level of health risk associated with pathogens in these environments. This review collaboratively focuses and summarizes the contamination levels of pathogens on the 5 water systems accessible to humans (natural water, drinking water, recreational water, wastewater, and reclaimed water). Then, we showcase the pathways, influencing factors and simulation models of pathogens transmission and survival. Further, we compare the health risk levels of various pathogens through Quantitative Microbial Risk Assessment (QMRA), and assess the limitations of water-associated QMRA application. Pathogen levels in wastewater are consistently higher than in other water systems, with no significant variation for Cryptosporidium spp. among five water systems. Hydraulic conditions primarily govern the transmission of pathogens into human-accessible waters, while environmental factors such as temperature impact pathogens survival. The median and mean values of computed public health risk levels posed by pathogens consistently surpass safety thresholds, particularly in the context of recreational waters. Despite the highest pathogens levels found in wastewater, the calculated health risk is significantly lower than in other water systems. Except pathogens concentration, variables like the exposure mode, extent, and frequency are also crucial factors influencing the public health risk in water systems. This review shares valuable insights to the more accurate assessment and comprehensive management of public health risk in human-accessible water environments.

Determining the Contribution of Micro/Nanoplastics to Antimicrobial Resistance: Challenges and Perspectives.

Microorganisms colonizing the surfaces of microplastics form a plastisphere in the environment, which captures miscellaneous substances. The plastisphere, owning to its inherently complex nature, may serve as a "Petri dish" for the development and dissemination of antibiotic resistance genes (ARGs), adding a layer of complexity in tackling the global challenge of both microplastics and ARGs. Increasing studies have drawn insights into the extent to which the proliferation of ARGs occurred in the presence of micro/nanoplastics, thereby increasing antimicrobial resistance (AMR). However, a comprehensive review is still lacking in consideration of the current increasingly scattered research focus and results. This review focuses on the spread of ARGs mediated by microplastics, especially on the challenges and perspectives on determining the contribution of microplastics to AMR. The plastisphere accumulates biotic and abiotic materials on the persistent surfaces, which, in turn, offers a preferred environment for gene exchange within and across the boundary of the plastisphere. Microplastics breaking down to smaller sizes, such as nanoscale, can possibly promote the horizontal gene transfer of ARGs as environmental stressors by inducing the overgeneration of reactive oxygen species. Additionally, we also discussed methods, especially quantitatively comparing ARG profiles among different environmental samples in this emerging field and the challenges that multidimensional parameters are in great necessity to systematically determine the antimicrobial dissemination risk in the plastisphere. Finally, based on the biological sequencing data, we offered a framework to assess the AMR risks of micro/nanoplastics and biocolonizable microparticles that leverage multidimensional AMR-associated messages, including the ARGs' abundance, mobility, and potential acquisition by pathogens.

Application of high-throughput sequencing technologies and analytical tools for pathogen detection in urban water systems: Progress and future perspectives.

The ubiquitous presence of pathogenic microorganisms, such as viruses, bacteria, fungi, and protozoa, in urban water systems poses a significant risk to public health. The emergence of infectious waterborne diseases mediated by urban water systems has become one of the leading global causes of mortality. However, the detection and monitoring of these pathogenic microorganisms have been limited by the complexity and diversity in the environmental samples. Conventional methods were restricted by long assay time, high benchmarks of identification, and narrow application sceneries. Novel technologies, such as high-throughput sequencing technologies, enable potentially full-spectrum detection of trace pathogenic microorganisms in complex environmental matrices. This review discusses the current state of high-throughput sequencing technologies for identifying pathogenic microorganisms in urban water systems with a concise summary. Furthermore, future perspectives in pathogen research emphasize the need for detection methods with high accuracy and sensitivity, the establishment of precise detection standards and procedures, and the significance of bioinformatics software and platforms. We have compiled a list of pathogens analysis software/platforms/databases that boast robust engines and high accuracy for preference. We highlight the significance of analyses by combining targeted and non-targeted sequencing technologies, short and long reads technologies, sequencing technologies, and bioinformatic tools in pursuing upgraded biosafety in urban water systems.

Microplastics extraction from wastewater treatment plants: Two-step digestion pre-treatment and application.

As the gathering place of urban wastewater, wastewater treatment plants (WWTPs) are indispensable for removing microplastics (MPs), one of the emerging contaminants of great concern, from cities into the natural environment. A reliable and efficient extraction method for MPs, especially in organic-rich matrices, such as sludge samples, is the basis for studying MPs contamination, while it is still lacking. The digestion process, which requires further optimisation, is the most important step during extraction. In this study, we developed and optimised a two-step digestion process to extract MPs and proposed a recommended dosage of digestion reagents based on the mixed liquid volatile suspended solids (MLVSS) level of the sample. Successive addition of 30% H2O2 + 1 M HNO3 (v:v = 1:1, T = 60 °C, t = 5 h + 5 h) could efficiently extract MPs from sludge samples (over 90%), and the recommended dosage of digestion reagent was 100 ml 30% H2O2+100 ml 1 M HNO3 with the sample MLVSS lower than approximately 0.43 g. This new method was also applied to examine the characteristics of MPs in two typical WWTPs (anaerobic-anoxic-oxic and biofilter processes) in Shenzhen. The concentrations of MPs in the influent, effluent and dewatered sludge were approximately 114.00 n/L, 6.00 n/L, and 126.00 n/g (dry weight) in WWTP A, whereas 404.00 n/L, 22.00 n/L, and 204.00 n/g (dry weight) in WWTP B, respectively. Rayon and polyester were the dominant polymers in both the WWTPs. Fibers accounted for the largest proportion of the influent and effluent. Sizes between 0.20-0.50 mm were most detected. This study provides a new and efficient reference method to extract MPs from WWTPs samples, especially sludge sample, with less MPs loss and more beneficial to subsequent identification.

Removing microplastics from aquatic environments: A critical review.

As one of the typical emerging contaminants, microplastics exist widely in the environment because of their small size and recalcitrance, which has caused various ecological problems. This paper summarizes current adsorption and removal technologies of microplastics in typical aquatic environments, including natural freshwater, marine, drinking water treatment plants (DWTPs), and wastewater treatment plants (WWTPs), and includes abiotic and biotic degradation technologies as one of the removal technologies. Recently, numerous studies have shown that enrichment technologies have been widely used to remove microplastics in natural freshwater environments, DWTPs, and WWTPs. Efficient removal of microplastics via WWTPs is critical to reduce the release to the natural environment as a key connection point to prevent the transfer of microplastics from society to natural water systems. Photocatalytic technology has outstanding pre-degradation effects on microplastics, and the isolated microbial strains or enriched communities can degrade up to 50% or more of pre-processed microplastics. Thus, more research focusing on microplastic degradation could be carried out by combining physical and chemical pretreatment with subsequent microbial biodegradation. In addition, the current recovery technologies of microplastics are introduced in this review. This is incredibly challenging because of the small size and dispersibility of microplastics, and the related technologies still need further development. This paper will provide theoretical support and advice for preventing and controlling the ecological risks mediated by microplastics in the aquatic environment and share recommendations for future research on the removal and recovery of microplastics in various aquatic environments, including natural aquatic environments, DWTPs, and WWTPs.

Distinct responses of Pseudomonas aeruginosa PAO1 exposed to different levels of polystyrene nanoplastics.

Large amounts of discarded plastics in the environment can be aged into microplastics and nanoplastics, which are not easily removed, posing potential nonnegligible risks to the ecosystem and human health. Although previous studies have revealed that nanoplastics have detrimental impacts on microorganisms, the potential molecular mechanisms of nanoplastic particles' effect on microbial growth and metabolism are still lacking. Here, multiple responses of Pseudomonas aeruginosa PAO1 (PAO1) to different levels of polystyrene nanoplastics (PS NPs) exposure were investigated by physiological experiments, live/dead staining, redox status, and genome-wide RNA sequencing. The results showed that PS NPs had dual effects on PAO1, and different concentrations of PS NPs demonstrated different effects on the growth and metabolism of PAO1. All levels of PS NPs had no obvious biocidal effect on PAO1. The production and consumption of ROS were in dynamic equilibrium and could be regulated genetically to ensure that the ROS level was in the biotolerable range. 20 and 50 mg/L of PS NPs severely inhibited the nitrate reduction, while 0.1 mg/L of PS NPs promoted the denitrification and TCA cycle. Meanwhile, 20 and 50 mg/L of PS NPs resulted in intense down-regulation of genes involved in denitrification. In contrast, the expression of genes involved in respiration is promoted with generated energy to withstand stress from high-level PS NPs, coinciding with the physiological results. In addition, our results showed that PS NPs concentrations of 20 and 50 mg/L exposure substantially up-regulated the expression of genes encoding for flagellar biosynthesis and biofilm formation to tackle the stress. Our findings would provide new insights into the interactions between environmental bacteria and PS NPs at the transcriptional level, thereby enhancing our understanding of the potential risks of PS NPs to microbial ecosystems and public health.

The contamination of microplastics in China's aquatic environment: Occurrence, detection and implications for ecological risk.

The widespread occurrence of microplastics in aquatic ecosystems that resulted in environmental contamination has attracted worldwide attention. Microplastics pose a potential threat to the growth and health of aquatic organisms, thereby affecting the function of the ecosystems. As one of the top ten countries producing and consuming plastic products globally, China's aquatic ecosystems have been profoundly affected by microplastics. In this review, we have summarized the microplastics contamination in three typical water environments (marine environment, freshwater environment, and wastewater treatment plants) in China, elaborated on the adverse impacts of microplastics on the ecological environment, and evaluated the potential ecological risks exposed to the ecosystem. In addition, the progress of microplastics extraction methods, as the important basis of microplastics related research, in aquatic ecosystems was introduced, especially the difference between the extraction of microplastics from wastewater and sludge samples. At present, most of the research on microplastics focuses on "one point", such as a certain river or wastewater treatment plant. Research on the mitigation and transfer of microplastics among different connected water environments is still lacking. Also, the microscale ecotoxicity caused by microplastics is poorly understood. In the end, we proposed suggestions and perspectives for future research regarding microplastics in the aquatic ecosystems in China.

Microbial Interactions Drive the Complete Catabolism of the Antibiotic Sulfamethoxazole in Activated Sludge Microbiomes.

Microbial communities are believed to outperform monocultures in the complete catabolism of organic pollutants via reduced metabolic burden and increased robustness to environmental challenges; however, the interaction mechanism in functional microbiomes remains poorly understood. Here, three functionally differentiated activated sludge microbiomes (S1: complete catabolism of sulfamethoxazole (SMX); S2: complete catabolism of the phenyl part of SMX ([phenyl]-SMX) with stable accumulation of its heterocyclic product 3-amino-5-methylisoxazole (3A5MI); A: complete catabolism of 3A5MI rather than [phenyl]-SMX) were enriched. Combining time-series cultivation-independent microbial community analysis, DNA-stable isotope probing, molecular ecological network analysis, and cultivation-dependent function verification, we identified key players involved in the SMX degradation process. Paenarthrobacter and Nocardioides were primary degraders for the initial cleavage of the sulfonamide functional group (-C-S-N- bond) and 3A5MI degradation, respectively. Complete catabolism of SMX was achieved by their cross-feeding. The co-culture of Nocardioides, Acidovorax, and Sphingobium demonstrated that the nondegraders Acidovorax and Sphingobium were involved in the enhancement of 3A5MI degradation. Moreover, we unraveled the internal labor division patterns and connections among the active members centered on the two primary degraders. Overall, the proposed methodology is promisingly applicable and would help generate mechanistic, predictive, and operational understanding of the collaborative biodegradation of various contaminants. This study provides useful information for synthetic activated sludge microbiomes with optimized environmental functions.

Microalgae harvesting by magnetic flocculation for biodiesel production: current status and potential.

With the increasing demand for energy, microalgae, as one of the promising feedstocks of biodiesel, has raised great awareness. Because of its small size, similar density to water and electrical stability, harvesting methods of microalgae that have low energy consumption and that are highly efficient, easy to large-scale and environmentally friendly have become a bottleneck restricting development of the whole process. Among the numerous possible harvesting methods, magnetic flocculation has the advantages of simple operation, fast separation and energy saving and thus is considered as a promising novel harvesting method. In this review, we have summarized the updated status and application potential of magnetic flocculation, including the principle of magnetic flocculation, magnetic flocculating materials, flocculating efficiency and its effect on downstream process. The major challenges such as magnetic materials recovery, large-scale magnetic flocculation device design, and magnetic flocculation costs are also discussed.

A Ruthenium Nitrosyl-Functionalized Magnetic Nanoplatform with Near-Infrared Light-Controlled Nitric Oxide Delivery and Photothermal Effect for Enhanced Antitumor and Antibacterial Therapy.

Developing a spatiotemporal-controlled nitric oxide (NO) delivery nanoplatform is highly desirable for its biological applications as a tumor inhibitor and antibacterial agent. In this study, a novel multifunctional magnetic nanoplatform {Fe3O4@PDA@Ru-NO@FA} (1) was developed for the near-infrared (NIR) light-controlled release of NO in which a ruthenium nitrosyl (Ru-NO) donor and a folic acid (FA)-directing group were covalently functionalized onto Fe3O4@PDA. Nanoplatform 1 preferentially accumulated in folate receptor-overexpressing cancer cell lines and magnetic field-guided tumor tissue, instantly released NO, and simultaneously produced a prominent photothermal effect upon 808 nm NIR light irradiation, leading to remarkable in vitro and in vivo antitumor efficacy. When nanoplatform 1 was treated only once, the potential MRI contrast agent was sufficient to significantly inhibit or eliminate the tumor tissues in living mice, thus offering opportunities for future NO-involved multimodal cancer therapy. In addition, a NO delivery nanoplatform {Fe3O4@PDA@Ru-NO} was imbedded in the matrix of a chitosan (CS)-poly(vinyl alcohol) (PVA) material to develop a hybrid thermosensitive CS-PVA/NO hydrogel. The CS-PVA/NO hydrogels demonstrated mild (<150 mW cm-2) NIR light-controlled NO delivery and thus produced an efficient antibacterial effect for both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Therefore, these hydrogels have potential as antibacterial dressings for wound bacterial infection treatment.

Screening and mutagenesis of high-efficient degrading bacteria of linear alkylbenzene sulfonates.

As a widely used detergent, anionic surfactant linear alkylbenzene sulfonates (LAS) is a common toxic pollutant in wastewater. In this study, Pseudomonas sp. strain H6 was isolated from activated sludge and municipal wastewater, which had good degradation effect on LAS. The results showed that strain H6 could grow with LAS as the sole carbon source. When the concentration of LAS was less than 100 mg/L, strain H6 could degrade more than 80% of the LAS within 24 h. Meanwhile, the growth of strain H6 increased with the increase of LAS concentration, reaching the maximum growth at the presence of 100 mg/L LAS. When the concentration of LAS was over 100 mg/L, strain H6's cell growth and degradation of LAS showed a downward trend due to the strong toxicity of LAS, and the degradation rate of LAS almost tended to zero with 500 mg/L LAS. Further mutagenesis analysis of strain H6 showed that positive mutation occurred under ultraviolet and nitrite mutagenesis with using ampicillin to increase the screening pressure, and the degradation rate of LAS was 44.91% higher than that of original strain.

Rapid enrichment and ammonia oxidation performance of ammonia-oxidizing archaea from an urban polluted river of China.

Ammonia oxidation is the rate-limiting step in nitrification process and dominated by ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). In the present study, a highly enriched culture of AOA was obtained from urban polluted water in Shahe River, Shenzhen, China. The optimum growth conditions were identified by orthogonal analysis as 37 °C, with pH 7.0 and initial ammonia concentration of 1.0 mM. Under these conditions, the highest abundance of AOA was obtained as 4.6 × 107 copies/ng DNA. Growth of AOA in polluted river water showed significant reduction in ammonia concentration in AOA-enriched cultures without antibiotics after 10 days of incubation, while synchronous increase in nitrate concentration was up to 12.7 mg/L. However, AOA-enriched by antibiotic showed insignificant changes in ammonia or nitrite concentration. This study showed that AOB play an important role in ammonia oxidation of polluted river water, and AOA alone showed insignificant changes in ammonia or nitrite concentrations. Therefore, the ammonia oxidation performance of natural water could not be improved by adding high concentration AOA bacterial liquid.

A comparative proteomic analysis of Desulfovibrio vulgaris Hildenborough in response to the antimicrobial agent free nitrous acid.

Sulfate reducing bacteria (SRB) can contribute to facilitating serious concrete corrosion through the production of hydrogen sulfide in sewers. Recently, free nitrous acid (FNA) was discovered as a promising antimicrobial agent to inhibit SRB activities thereby limiting hydrogen sulfide production in sewers. However, knowledge of the bacterial response to increasing levels of the antimicrobial agent is unknown. Here we report the proteomic response of Desulfovibrio vulgaris Hildenborough and reveal that the antimicrobial effect of FNA is multi-targeted and dependent on the FNA levels. This was achieved using a sequential window acquisition of all theoretical mass spectrometry analysis to determine protein abundance variations in D. vulgaris during exposure to different FNA concentrations. When exposed to 1.0 µg N/L FNA, nitrite reduction (nitrite reductase) related proteins and nitrosative stress related proteins, including the hybrid cluster protein, showed distinct increased abundances. When exposed to 4.0 and 8.0 µg N/L FNA, increased abundance was detected for proteins putatively involved in nitrite reduction. Abundance of proteins involved in the sulfate reduction pathway (from adenylylphophosulfate to sulfite) and lactate oxidation pathway (from pyruvate to acetate) were initially inhibited in response to FNA at 8 h incubation, and then recovered at 12 h incubation. Lowered ribosomal protein abundance in D. vulgaris was detected, however, total cellular protein levels were mostly constant in the presence or absence of FNA. In addition, this study indicates that proteins coded by genes DVU2543, DVU0772, and DVU3212 potentially participate in resisting oxidative stress with FNA exposure. These findings share new insights for understanding the dynamic responses of D. vulgaris to FNA and could be useful to guide and improve the practical applications of FNA-based technologies for control of sewer corrosion.

Physiological and transcriptomic analyses reveal CuO nanoparticle inhibition of anabolic and catabolic activities of sulfate-reducing bacterium.

The widespread use of CuO nanoparticles (NPs) results in their continuous release into the environment, which could pose risks to public health and to microbial ecosystems. Following consumption, NPs will initially enter into sewer systems and interact with and potentially influence sewer microbial communities. An understanding of the response of microbes in sewers, particularly sulfate-reducing bacteria (SRB), to the CuO NPs induced stress is important as hydrogen sulfide produced by SRB can cause sewer corrosion and odour emissions. In this study, we elucidated how the anabolic and catabolic processes of a model SRB, Desulfovibrio vulgaris Hidenborough (D. vulgaris), respond to CuO NPs. Physiological analyses indicated that the exposure of the culture to CuO NPs at elevated concentrations (>50 mg/L) inhibited both its anabolic and catabolic activities, as revealed by lowered cell proliferation and sulfate reduction rate. The antibacterial effects of CuO NPs were mainly attributed to the overproduction of reactive oxygen species. Transcriptomic analysis indicated that genes encoding for flagellar assembly and some genes involved in electron transfer and respiration were down-regulated, while genes for the ferric uptake regulator (Fur) were up-regulated. Moreover, the CuO NPs exposure significantly up-regulated genes involved in protein synthesis and ATP synthesis. These results suggest that CuO NPs inhibited energy conversion, cell mobility, and iron starvation to D. vulgaris. Meanwhile, D. vulgaris attempted to respond to the stress of CuO NPs by increasing protein and ATP synthesis. These findings offer new insights into the bacterial-nanoparticles interaction at the transcriptional level, and advance our understanding of impacts of CuO NPs on SRB in the environment.

Ubiquity of polystyrene digestion and biodegradation within yellow mealworms, larvae of Tenebrio molitor Linnaeus (Coleoptera: Tenebrionidae).

Academics researchers and "citizen scientists" from 22 countries confirmed that yellow mealworms, the larvae of Tenebrio molitor Linnaeus, can survive by eating polystyrene (PS) foam. More detailed assessments of this capability for mealworms were carried out by12 sources: five from the USA, six from China, and one from Northern Ireland. All of these mealworms digested PS foam. PS mass decreased and depolymerization was observed, with appearance of lower molecular weight residuals and functional groups indicative of oxidative transformations in extracts from the frass (insect excrement). An addition of gentamycin (30 mg g-1), a bactericidal antibiotic, inhibited depolymerization, implicating the gut microbiome in the biodegradation process. Microbial community analyses demonstrated significant taxonomic shifts for mealworms fed diets of PS plus bran and PS alone. The results indicate that mealworms from diverse locations eat and metabolize PS and support the hypothesis that this capacity is independent of the geographic origin of the mealworms, and is likely ubiquitous to members of this species.

Biodegradation of Polyethylene and Plastic Mixtures in Mealworms (Larvae of Tenebrio molitor) and Effects on the Gut Microbiome.

Recent studies have demonstrated the ability for polystyrene (PS) degradation within the gut of mealworms ( Tenebrio molitor). To determine whether plastics may be broadly susceptible to biodegradation within mealworms, we evaluated the fate of polyethylene (PE) and mixtures (PE + PS). We find that PE biodegrades at comparable rates to PS. Mass balances indicate conversion of up 49.0 ± 1.4% of the ingested PE into a putative gas fraction (CO2). The molecular weights ( Mn) of egested polymer residues decreased by 40.1 ± 8.5% in PE-fed mealworms and by 12.8 ± 3.1% in PS-fed mealworms. NMR and FTIR analyses revealed chemical modifications consistent with degradation and partial oxidation of the polymer. Mixtures likewise degraded. Our results are consistent with a nonspecific degradation mechanism. Analysis of the gut microbiome by next-generation sequencing revealed two OTUs ( Citrobacter sp. and Kosakonia sp.) strongly associated with both PE and PS as well as OTUs unique to each plastic. Our results suggest that adaptability of the mealworm gut microbiome enables degradation of chemically dissimilar plastics.

Subcritical n-hexane/isopropanol extraction of lipid from wet microalgal pastes of Scenedesmus obliquus.

Subcritical co-solvents of n-hexane/isopropanol were primarily utilized to extract lipid from wet microalgal pastes of Scenedesmus obliquus. The effects of key operational parameters were investigated, and the optimal parameters were obtained: solvent ratio of n-hexane to isopropanol was 3:2 (V:V), phase ratio of co-solvents to microalgal biomass was 35:1 (mL:g), reactor stirring speed was 900 rpm, extraction time was 60 min. Additional pretreatment with acid, ultrasonic and microwave as well as enhanced subcritical pressure/heating treatments were also applied to further study their effects on lipid extraction. The results showed that the lipid recovery rate with acid pretreatment was 8.6 and 6.2% higher than ultrasonic and microwave pretreatment; the optimum enhanced subcritical condition was 55 °C with atmospheric pressure. Under optimal operating conditions, the lipid and FAME yield were 13.5 and 7.2%, which was 82.6 and 135.1% higher than the traditional method. The results indicated that the subcritical n-hexane/isopropanol extraction process had promising application potential.