Bioaugmentation of anaerobic digesters with the enriched lignin-degrading microbial consortia through a metagenomic approach.

The recalcitrance of lignin impedes the efficient utilization of lignocellulosic biomass, hindering the efficient production of biogas and value-added materials. Despite the emergence of anaerobic digestion as a superior alternative to the aerobic method for lignin processing, achieving its feasibility requires thorough characterization of lignin-degrading anaerobic microorganisms, assessment of their biomethane production potential, and a comprehensive understanding of the degradation pathway. This study aimed to address the aforementioned necessities by bioaugmenting seed sludge with three distinct enriched lignin-degrading microbial consortia at both 25 °C and 37 °C. Enhanced biomethane yields was detected in the bioaugmented digesters, while the highest production was observed as 188 mLN CH4 gVS-1 in digesters operated at 37 °C. Moreover, methane yield showed a significant improvement in the samples at 37 °C ranging from 110% to 141% compared to the control, demonstrating the efficiency of the enriched lignin-degrading microbial community. Temperature and substrate were identified as key factors influencing microbial community dynamics. The observation that microbial communities tended to revert to the initial state after lignin depletion, indicating the stability of the overall microbiota composition in the digesters, is a promising finding for large-scale studies. Noteworthy candidates for lignin degradation, including Sporosarcina psychrophila, Comamonas aquatica, Shewanella baltica, Pseudomonas sp. C27, and Brevefilum fermentans were identified in the bioaugmented samples. PICRUSt2 predictions suggest that the pathway and specific proteins involved in anaerobic lignin degradation might share similarities with those engaged in the degradation of aromatic compounds.

Is a floodplain forest a valuable source for lignin-degrading anaerobic microbial communities: A metagenomic approach.

Lignin is one of the most substantial obstacles in the evaluation of lignocellulosic compounds. Although there are numerous approaches for the enhancement of lignin digestion in the literature, there has yet to be an optimized system to date. In this study, samples taken from Igneada floodplain forests were enriched anaerobically at 25 °C and 37 °C, with alkali lignin as the sole carbon source. The activity of the anaerobic lignin-degrading microbial consortium was detected more efficiently at 37 °C, where biogas production exceeded 3.5 mLgas/mLmedium. It was observed that the microbial community initially dominated by Proteobacteria (around 60%) changed completely after enrichment and was led by members of the Firmicutes phylum (up to 90%). The dominant species (Sporomusa termitida, Desulfitobacterium hafniense, Citrobacter freundii, Citrobacter portucalensis, Alkalibacter rhizosphaerae, and Gudongella oleilytica) occupying more than 50% in the final enrichment culture were only around 2% in the raw samples. Therefore, this study, one of the few in which enriched environmental samples were sequenced using MinION, demonstrated that longoses are exceptional reservoirs for lignin-digesting anaerobic microorganisms.

Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents.

AIMS: A new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction. METHODS: Spiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 L FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA). RESULTS: No qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%-100% (69/72-72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA. CONCLUSIONS: The developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.

Lab-on-a-chip systems for cancer biomarker diagnosis.

Lab-on-a-chip (LOC) or micro total analysis system is one of the microfluidic technologies defined as the adaptation, miniaturization, integration, and automation of analytical laboratory procedures into a single instrument or "chip". In this article, we review developments over the past five years in the application of LOC biosensors for the detection of different types of cancer. Microfluidics encompasses chemistry and biotechnology skills and has revolutionized healthcare diagnosis. Superior to traditional cell culture or animal models, microfluidic technology has made it possible to reconstruct functional units of organs on chips to study human diseases such as cancer. LOCs have found numerous biomedical applications over the past five years, including integrated bioassays, cell analysis, metabolomics, drug discovery and delivery systems, tissue and organ physiology and disease modeling, and personalized medicine. This review provides an overview of the latest developments in microfluidic-based cancer research, with pros, cons, and prospects.

Fluid-based wearable sensors: a turning point in personalized healthcare.

Nowadays, it has become very popular to develop wearable devices that can monitor biomarkers to analyze the health status of the human body more comprehensively and accurately. Wearable sensors, specially designed for home care services, show great promise with their ease of use, especially during pandemic periods. Scientists have conducted many innovative studies on new wearable sensors that can noninvasively and simultaneously monitor biochemical indicators in body fluids for disease prediction, diagnosis, and management. Using noninvasive electrochemical sensors, biomarkers can be detected in tears, saliva, perspiration, and skin interstitial fluid (ISF). In this review, biofluids used for noninvasive wearable sensor detection under four main headings, saliva, sweat, tears, and ISF-based wearable sensors, were examined in detail. This report analyzes nearly 50 recent articles from 2017 to 2023. Based on current research, this review also discusses the evolution of wearable sensors, potential implementation challenges, and future prospects.

Lateral flow assays for viruses diagnosis: Up-to-date technology and future prospects.

Bacteria, viruses, and parasites are harmful microorganisms that cause infectious diseases. Early detection of diseases is critical to prevent disease transmission and provide epidemic preparedness, as these can cause widespread deaths and public health crises, particularly in resource-limited countries. Lateral flow assay (LFA) systems are simple-to-use, disposable, inexpensive diagnostic devices to test biomarkers in blood and urine samples. Thus, LFA has recently received significant attention, especially during the pandemic. Here, first of all, the design principles and working mechanisms of existing LFA methods are examined. Then, current LFA implementation strategies are presented for communicable disease diagnoses, including COVID-19, zika and dengue, HIV, hepatitis, influenza, malaria, and other pathogens. Furthermore, this review focuses on an overview of current problems and accessible solutions in detecting infectious agents and diseases by LFA, focusing on increasing sensitivity with various detection methods. In addition, future trends in LFA-based diagnostics are envisioned.

Biosensing strategies for diagnosis of prostate specific antigen.

Almost from the time of its discovery, the prostate specific antigen (PSA) has been one of the most accurate and most extensively studied indicators of prostate cancer (PC). Because of advancements in biosensing systems and technology, PSA analysis methods have been substantially updated and enhanced as compared to their first instances. With the development of techniques in biosensor technology, the number of PSA biosensors that can be used in the biomedical sector is increasing year by year. Many different recognition elements and transducers have been used in the development of biosensor systems that exhibit high sensitivity, selectivity, and specificity. Here in this review, we provide a current overview of the different approaches to PSA detection.

Crop-based composting of lignocellulosic digestates: Focus on bacterial and fungal diversity.

In this study, organic matter degradation and microbial diversity were assessed during the composting of lignocellulose-rich digestates. Digestates were collected based on each crop type during anaerobic co-digestion of cow manure and barley, triticale, wheat and rye. Bacterial and fungal diversity in digestate composting systems were determined by 16S and 18S rRNA gene amplicon sequencing, respectively. Crop-based composting of anaerobic digestates showed similar process trends in terms of pH, temperature, moisture content (MC) and C:N ratio. The properties of final compost products were in accordance with the national legislations regarding soil applications, except MC, which were therefore air-dried before being amended to soil. Most abundant bacterial genera were represented by Luteimonas, Bacillus, Ochrobactrum and Thermobifida. Meanwhile, Thermomyces, Aspergillus, Galactomyces and Neurospora were detected as the predominant fungal genera in all compost samples.

Linking nano-ZnO contamination to microbial community profiling in sanitary landfill simulations.

Nanomaterials (NMs) commercially used for various activities mostly end up in landfills. Reduced biogas productions reported in landfill reactors create a need for more comprehensive research on these greatly-diverse microbial pools. In order to evaluate the impact of one of the most widely-used NMs, namely nano-zinc oxide (nano-ZnO), simulated bioreactor and conventional landfills were operated using real municipal solid waste (MSW) for 300 days with addition nano-ZnO. Leachate samples were taken at different phases and analyzed by 16S rRNA gene amplicon sequencing. The bacterial communities were distinctly characterized by Cloacamonaceae (phylum WWE1), Rhodocyclaceae (phylum Proteobacteria), Porphyromonadaceae (phylum Bacteroidetes), and Synergistaceae (phylum Synergistetes). The bacterial community in the bioreactors shifted at the end of the operation and was dominated by Rhodocyclaceae. There was not a major change in the bacterial community in the conventional reactors. The methanogenic archaeal diversity highly differed between the bioreactors and conventional reactors. The dominance of Methanomicrobiaceae was observed in the bioreactors during the peak methane-production period; however, their prominence shifted to WSA2 in the nano-ZnO-added bioreactor and to Methanocorpusculaceae in the control bioreactor towards the end. Methanocorpusculaceae was the most abundant family in both conventional control and nano-ZnO-containing reactors.

Fungal bioaugmentation of anaerobic digesters fed with lignocellulosic biomass: What to expect from anaerobic fungus Orpinomyces sp.

Energy-efficient biogas reactors are often designed and operated mimicking natural microbial ecosystems such as the digestive tracts of ruminants. Anaerobic fungi play a crucial role in the degradation of lignocellulose-rich fiber thanks to their high cellulolytic activity. Fungal bioaugmentation is therefore at the heart of our understanding of enhancing anaerobic digestion (AD). The efficiency of bioaugmentation with anaerobic fungus Orpinomyces sp. was evaluated in lignocellulose-based AD configurations. Fungal bioaugmentation increased the methane yield by 15-33% during anaerobic co-digestion of cow manure and selected cereal crops/straws. Harvesting stage of the crops was a decisive parameter to influence methane production together with fungal bioaugmentation. A more efficient fermentation process in the bioaugmented digesters was distinguished by relatively-higher abundance of Synergistetes, which was mainly represented by the genus Anaerobaculum. On the contrary, the composition of the methanogenic archaea did not change, and the majority of methanogens was assigned to Methanosarcina.

Comparison of Rumen and Manure Microbiomes and Implications for the Inoculation of Anaerobic Digesters.

Cattle manure is frequently used as an inoculum for the start-up of agricultural biogas plants or as a co-substrate in the anaerobic digestion of lignocellulosic feedstock. Ruminal microbiota are considered to be effective plant fiber degraders, but the microbes contained in manure do not necessarily reflect the rumen microbiome. The aim of this study was to compare the microbial community composition of cow rumen and manure with respect to plant fiber-digesting microbes. Bacterial and methanogenic communities of rumen and manure samples were examined by 454 amplicon sequencing of bacterial 16S rRNA genes and mcrA genes, respectively. Rumen fluid samples were dominated by Prevotellaceae (29%), whereas Ruminococcaceae was the most abundant family in the manure samples (31%). Fibrobacteraceae (12%) and Bacteroidaceae (13%) were the second most abundant families in rumen fluid and manure, respectively. The high abundances of fiber-degrading bacteria belonging to Prevotellaceae and Fibrobacteraceae might explain the better performance of anaerobic digesters inoculated with rumen fluid. Members of the genus Methanobrevibacter were the predominant methanogens in the rumen fluid, whereas methanogenic communities of the manure samples were dominated by the candidate genus Methanoplasma. Our results suggest that inoculation or bioaugmentation with fiber-digesting rumen microbiota can enhance the anaerobic digestion of lignocellulosic biomass.

Enrichment of lignocellulose-degrading microbial communities from natural and engineered methanogenic environments.

The aim of this study was to develop an effective bioaugmentation concept for anaerobic digesters treating lignocellulosic biomass such as straw. For that purpose, lignocellulose-degrading methanogenic communities were enriched on wheat straw from cow and goat rumen fluid as well as from a biogas reactor acclimated to lignocellulosic biomass (sorghum as mono-substrate). The bacterial communities of the enriched cultures and the different inocula were examined by 454 amplicon sequencing of 16S rRNA genes while the methanogenic archaeal communities were analyzed by terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the mcrA gene. Bacteroidetes was the most abundant phylum in all samples. Within the Bacteroidetes phylum, Bacteroidaceae was the most abundant family in the rumen-derived enrichment cultures, whereas Porphyromonadaceae was the predominant one in the reactor-derived culture. Additionally, the enrichment procedure increased the relative abundance of Ruminococcaceae (phylum: Firmicutes) in all cultures. T-RFLP profiles of the mcrA gene amplicons highlighted that the ruminal methanogenic communities were composed of hydrogenotrophic methanogens dominated by the order Methanobacteriales regardless of the host species. The methanogenic communities changed significantly during the enrichment procedure, but still the strict hydrogenotrophic Methanobacteriales and Methanomicrobiales were the predominant orders in the enrichment cultures. The bioaugmentation potential of the enriched methanogenic cultures will be evaluated in further studies.

Bioaugmentation with Clostridium thermocellum to enhance the anaerobic biodegradation of lignocellulosic agricultural residues.

This study aimed to improve biomethane production from lignocellulosic biomass by assessing the impact of bioaugmentation with Clostridium thermocellum on the performance of anaerobic digesters at different inoculation ratios. The outputs of the digestion experiments revealed that bioaugmentation strategies with C. thermocellum increased the methane yield up to 39%. The sequencing analysis indicated that the indigenous microbial community was modified by the bioaugmentation. During the process of bioaugmentation, in the digester that was inoculated at the ratio of 20% (v:v), an increase in the abundance of Ruminococcaceae family led to a decrease in the Bacteroidaceae and Synergistaceae families. Furthermore, the metabolic products of the bioaugmented strains greatly influenced the diversity of the archaeal community and an increase in the abundance of Methanomicrobiales was observed.

Bioaugmentation of anaerobic digesters treating lignocellulosic feedstock by enriched microbial consortia.

Three different bioaugmentation cultures enriched from natural and engineered cellulolytic environments (cow and goat rumen, a biogas reactor digesting sorghum biomass) were compared for their enhancement potential on the anaerobic digestion of wheat straw. Methane yields were determined in batch tests using the Automatic Methane Potential Test System operated for 30 days under mesophilic conditions. All cultures had positive effects on substrate degradation, and higher methane yields were observed in the bioaugmented reactors compared to control reactors set up with standard inoculum. However, the level of enhancement differed according to the type of the enrichment culture. Methane yield in batch reactors augmented with 2% cow rumen derived enrichment culture was increased by only 6%. In contrast, reactors amended with 2% goat rumen derived enrichment culture or with the bioaugmentation culture obtained from the biogas reactor digesting sorghum biomass produced 27 and 20% more methane, respectively. The highest methane yield was recorded in reactors amended with 6% goat rumen derived enrichment culture, which represented an increase by 36%. The microbial communities were quite similar at the end of the batch tests independently of the bioaugmentation sources, indicating that the introduced microbial communities of the enrichment cultures did not dominate the reactors.

Operating conditions influence microbial community structures, elimination of the antibiotic resistance genes and metabolites during anaerobic digestion of cow manure in the presence of oxytetracycline.

The way that antibiotic residues in manure follow is one of the greatest concerns due to its potential negative impacts on microbial communities, the release of metabolites and antibiotic resistant genes (ARGs) into the nature and the loss of energy recovery in anaerobic digestion (AD) systems. This study evaluated the link between different operating conditions, the biodegradation of oxytetracycline (OTC) and the formation of its metabolites and ARGs in anaerobic digesters treating cow manure. Microbial communities and ARGs were determined through the use of quantitative real-time PCR. The biodegradation of OTC and occurrence of metabolites were determined using UV-HPLC and LC/MS/MS respectively. The maximum quantity of resistance genes was also examined at the beginning of AD tests and concentration was in the order of: tetM >tetO. The numbers of ARGs were always higher at high volatile solids (VS) content and high mixing rate. The results of the investigation revealed that relationship between mixing rate and VS content plays a crucial role for elimination of ARGs, OTC and metabolites. This can be attributed to high abundance of microorganisms due to high VS content and their increased contact with elevated mixing rate. An increased interaction between microorganisms triggers the promotion of ARGs.

Application of next-generation sequencing methods for microbial monitoring of anaerobic digestion of lignocellulosic biomass.

The anaerobic digestion of lignocellulosic wastes is considered an efficient method for managing the world's energy shortages and resolving contemporary environmental problems. However, the recalcitrance of lignocellulosic biomass represents a barrier to maximizing biogas production. The purpose of this review is to examine the extent to which sequencing methods can be employed to monitor such biofuel conversion processes. From a microbial perspective, we present a detailed insight into anaerobic digesters that utilize lignocellulosic biomass and discuss some benefits and disadvantages associated with the microbial sequencing techniques that are typically applied. We further evaluate the extent to which a hybrid approach incorporating a variation of existing methods can be utilized to develop a more in-depth understanding of microbial communities. It is hoped that this deeper knowledge will enhance the reliability and extent of research findings with the end objective of improving the stability of anaerobic digesters that manage lignocellulosic biomass.

Microbial monitoring of ammonia removal in a UASB reactor treating pre-digested chicken manure with anaerobic granular inoculum.

Performance and microbial community dynamics in an upflow anaerobic sludge bed (UASB) reactor coupled with anaerobic ammonium oxidizing (Anammox) treating diluted chicken manure digestate (Total ammonia nitrogen; TAN=123±10mg/L) were investigated for a 120-d operating period in the presence of anaerobic granular inoculum. Maximum TAN removal efficiency reached to above 80% with as low as 20mg/L TAN concentrations in the effluent. Moreover, total COD (tCOD) with 807±215mg/L in the influent was removed by 60-80%. High-throughput sequencing revealed that Proteobacteria, Actinobacteria, and Firmicutes were dominant phyla followed by Euryarchaeota and Bacteroidetes. The relative abundance of Planctomycetes significantly increased from 4% to 8-9% during the late days of the operation with decreased tCOD concentration, which indicated a more optimum condition to favor ammonia removal through anammox route. There was also significant association between the hzsA gene and ammonia removal in the UASB reactor.

Assessment of the horizontal transfer of functional genes as a suitable approach for evaluation of the bioremediation potential of petroleum-contaminated sites: a mini-review.

Petroleum sludge contains recalcitrant residuals. These compounds because of being toxic to humans and other organism are of the major concerns. Therefore, petroleum sludge should be safely disposed. Physicochemical methods which are used by this sector are mostly expensive and need complex devices. Bioremediation methods because of being eco-friendly and cost-effective overcome most of the limitations of physicochemical treatments. Microbial strains capable to degrade petroleum hydrocarbons are practically present in all soils and sediments and their population density increases in contact with contaminants. Bacterial strains cannot degrade alone all kinds of petroleum hydrocarbons, rather microbial consortium should collaborate with each other for degradation of petroleum hydrocarbon mixtures. Horizontal transfer of functional genes between bacteria plays an important role in increasing the metabolic potential of the microbial community. Therefore, selecting a suitable degrading gene and tracking its horizontal transfer would be a useful approach to evaluate the bioremediation process and to assess the bioremediation potential of contaminated sites.

Effect of bioaugmentation by cellulolytic bacteria enriched from sheep rumen on methane production from wheat straw.

The aim of this study was to determine the potential of bioaugmentation with cellulolytic rumen microbiota to enhance the anaerobic digestion of lignocellulosic feedstock. An anaerobic cellulolytic culture was enriched from sheep rumen fluid using wheat straw as substrate under mesophilic conditions. To investigate the effects of bioaugmentation on methane production from straw, the enrichment culture was added to batch reactors in proportions of 2% (Set-1) and 4% (Set-2) of the microbial cell number of the standard inoculum slurry. The methane production in the bioaugmented reactors was higher than in the control reactors. After 30 days of batch incubation, the average methane yield was 154 mLN CH4 gVS-1 in the control reactors. Addition of 2% enrichment culture did not enhance methane production, whereas in Set-2 the methane yield was increased by 27%. The bacterial communities were examined by 454 amplicon sequencing of 16S rRNA genes, while terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of mcrA genes was applied to analyze the methanogenic communities. The results highlighted that relative abundances of Ruminococcaceae and Lachnospiraceae increased during the enrichment. However, Cloacamonaceae, which were abundant in the standard inoculum, dominated the bacterial communities of all batch reactors. T-RFLP profiles revealed that Methanobacteriales were predominant in the rumen fluid, whereas the enrichment culture was dominated by Methanosarcinales. In the batch rectors, the most abundant methanogens were affiliated to Methanobacteriales and Methanomicrobiales. Our results suggest that bioaugmentation with sheep rumen enrichment cultures can enhance the performance of digesters treating lignocellulosic feedstock.

Development of a fast and low-cost qPCR assay for diagnosis of acute gas pharyngitis.

BACKGROUND: Group A streptococci (GAS) are the most common bacterial cause of acute pharyngitis and account for 15-30 % of cases of acute pharyngitis in children and 5-10 % of cases in adults. In this study, a real-time quantitative PCR (qPCR) based GAS detection assay in pharyngeal swab specimens was developed. METHODS: The qPCR assay was compared with the gold standard bacterial culture and a rapid antigen detection test (RADT) to evaluate its clinical performance in 687 patients. The analytical sensitivity of the assay was 240 cfu/swab. Forty-five different potential cross-reacting organisms did not react with the test. Four different laboratories for the reproducibility studies were in 100 % (60/60) agreement for the contrived GAS positive and negative swab samples. RESULTS: The relative sensitivities of the RADT and the qPCR test were 55.9 and 100 %; and the relative specificities were 100 and 96.3 %, respectively. Duration of the total assay for 24 samples including pre-analytical processing and analysis changed between 42 and 55 min depending on the type of qPCR instrument used. A simple DNA extraction method and a low qPCR volume made the developed assay an economical alternative for the GAS detection. CONCLUSION: We showed that the developed qPCR test is rapid, cheap, sensitive and specific and therefore can be used to replace both antigen detection and culture for diagnosis of acute GAS pharyngitis.