Enrichment Culture Research Articles

Probiotic yoghurt-like fermented milk product enriched with Lactobacillus desidiosus and Lactobacillus fermentum: proximate composition, physicochemical, microbiological, and sensory evaluation during refrigerated storage

The combination of multiple strains and multiple species in lactic acid bacteria-based fermented milk offers the potential for nutritional and sensory attributes, making it an attractive option for developing high-quality and health-promoting probiotic yoghurt-like fermented milk products. In the present study, four combinations of yoghurt-like fermented milk samples: Sample 1 (Control), Sample 2 (Lactobacillus desidiosus), Sample 3 (Lactobacillus fermentum), and Sample 4 (Lactobacillus desidiosus and Lactobacillus fermentum) were developed. Proximate composition (moisture, ash, protein, fat, carbohydrate), physicochemical (pH, titratable acidity, syneresis, total soluble sloid), microbiological (Lactobacillus bacteria, yeast and mold, and coliform bacteria), and sensory properties (color, flavor, texture, taste, and overall acceptability) were assessed under refrigerator conditions (4 °C) from 0 to 28 days of storage period. Proximate composition differed significantly (p < 0.05) among samples for moisture (49.34–79.32%), ash (1.14–0.31%), protein (3.72–3.21%), fat (11.23–5.62%), and carbohydrate (33.11–12.98%). Yoghurt-like fermented milk containing Lactobacillus desidiosus as single or in combination with Lactobacillus fermentum resulted in reduced pH levels (6.02–3.49), total soluble solid (41.66–24.66%) and increased syneresis (30.04–65.52%), titratable acidity content (0.42–1.62%). Single or combination of Lactobacillus desidiosus and Lactobacillus fermentum significantly (p < 0.05) reduced Lactobacillus bacteria (8.17–6.01 Log CFU/g), yeast, and mold (1.45–1.19 Log CFU/g). Additionally, none of the samples of probiotic yoghurt-like fermented milk showed any detectable coliform count, indicating the possibility of prolonging the shelf life. The sample prepared by Lactobacillus desidiosus had the highest sensory ranking (8.10) at day 0 and during storage than other samples. In conclusion, the inclusion of Lactobacillus desidiosus and Lactobacillus fermentum strains in the composition of production starter cultures holds great promise. The utilization of these enriched starter cultures enables the production of healthy food products that maintain their quality throughout the entire storage period.

Uncovering Hidden Microbial Diversity in Nitrate/Iodide Deposits (NIDs) in the Domeyko District, Atacama Desert, Chile

Unique worldwide, nitrate/iodine deposits (NIDs) are located along a 700 km geological belt in the Atacama Desert, Chile. They serve as the primary source of mineral ores for the extraction of iodine, sodium, and potassium nitrates. NIDs have been relatively underexplored from a biological perspective. To address this, we collected sixteen soil samples from abandoned mines in Oficinas Pissis and Savona for chemical, mineralogical, and metagenomic analyses. The soils primarily consisted of halite and darapskite, with only one sample being predominantly composed of thenardite. Deliquescence and water activity measurements yielded values ranging from 0.02% to 0.40% and 0.47 to 0.62, respectively. To investigate the presence, identification, relative abundance, and diversity of microbial life in NID soils, we employed MiSeq high-throughput sequencing and bioinformatic tools. The dominant phyla observed were Firmicutes and Proteobacteria, with Actinobacteria and Cyanobacteria being predominant in two soil samples. Furthermore, we detected nitrate/perchlorate-reducing bacterial activity in enriched cultures from the soil samples. This study sheds light on the resilience of microbial life in the Atacama Desert NIDs, providing compelling evidence for its existence and offering insight into factors that could facilitate it within this unique environment.

Low-volume enrichment method supports high throughput bacteriophage screening and isolation from wastewater.

Bacteriophage therapy is a rapidly growing field of study. Narrow host ranges, bacterial resistance, and limited antibiotic availability make lytic phages a feasible therapeutic potential. Phage discovery, a critical step in developing phage therapy, is a pathway to accessible treatment. This has always been a laborious, time-consuming and resource-intensive process. In this paper, we describe a 96-well plate low-volume bacteriophage enrichment method with concentrated environmental sources to rapidly discover and isolate phages targeting multiple organisms simultaneously. Samples from natural water sources, wastewater influent, and activated sludge were tested in large volume enrichment cultures and low-volume 96-well plate format. Each plate has the capacity to run as many as 48 different combinations with multiple bacterial hosts. The time to identify the presence of phage in a sample was 5 to 10 hours in the low-volume format versus a minimum of 2 days in the traditional enrichment method. The labor and expense involved also favor the 96-well plate format. There was some loss of discovered phages using this technique, primarily targeting bacterial species less prevalent in the environment. This is an easily modifiable method that is amenable to automation and a variety of potential phage sources.

Phenotypic and genomic characterization of Methanothermobacter wolfeii strain BSEL, a CO2-capturing archaeon with minimal nutrient requirements.

A new variant of Methanothermobacter wolfeii was isolated from an anaerobic digester using enrichment cultivation in anaerobic conditions. The new isolate was taxonomically identified via 16S rRNA gene sequencing and tagged as M. wolfeii BSEL. The whole genome of the new variant was sequenced and de novo assembled. Genomic variations between the BSEL strain and the type strain were discovered, suggesting evolutionary adaptations of the BSEL strain that conferred advantages while growing under a low concentration of nutrients. M. wolfeii BSEL displayed the highest specific growth rate ever reported for the wolfeii species (0.27 ± 0.03 h-1) using carbon dioxide (CO2) as unique carbon source and hydrogen (H2) as electron donor. M. wolfeii BSEL grew at this rate in an environment with ammonium (NH4+) as sole nitrogen source. The minerals content required to cultivate the BSEL strain was relatively low and resembled the ionic background of tap water without mineral supplements. Optimum growth rate for the new isolate was observed at 64°C and pH 8.3. In this work, it was shown that wastewater from a wastewater treatment facility can be used as a low-cost alternative medium to cultivate M. wolfeii BSEL. Continuous gas fermentation fed with a synthetic biogas mimic along with H2 in a bubble column bioreactor using M. wolfeii BSEL as biocatalyst resulted in a CO2 conversion efficiency of 97% and a final methane (CH4) titer of 98.5%v, demonstrating the ability of the new strain for upgrading biogas to renewable natural gas.IMPORTANCEAs a methanogenic archaeon, Methanothermobacter wolfeii uses CO2 as electron acceptor, producing CH4 as final product. The metabolism of M. wolfeii can be harnessed to capture CO2 from industrial emissions, besides producing a drop-in renewable biofuel to substitute fossil natural gas. If used as biocatalyst in new-generation CO2 sequestration processes, M. wolfeii has the potential to accelerate the decarbonization of the energy generation sector, which is the biggest contributor of CO2 emissions worldwide. Nonetheless, the development of CO2 sequestration archaeal-based biotechnology is still limited by an uncertainty in the requirements to cultivate methanogenic archaea and the unknown longevity of archaeal cultures. In this study, we report the adaptation, isolation, and phenotypic characterization of a novel variant of M. wolfeii, which is capable of maximum growth with minimal nutrients input. Our findings demonstrate the potential of this variant for the production of renewable natural gas, paving the way for the development of more efficient and sustainable CO2 sequestration processes.

In silico and PCR Screening for a Live Attenuated Salmonella Typhimurium Vaccine Strain.

The application of live attenuated Salmonella Typhimurium vaccines has significantly helped control Salmonella in poultry products. Because the U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) scores all Salmonella as positive, regardless of serovar, attenuated vaccine strains that are identified at processing contribute negatively toward Salmonella performance standards. This study was designed to determine the incidence of a live attenuated Salmonella serovar Typhimurium vaccine identified in broiler products by FSIS and to develop a PCR assay for screening of isolates. Salmonella Typhimurium short-read sequences from broiler samples uploaded to the National Center for Biotechnology Information (NCBI) Pathogen Detection database by the USDA-FSIS from 2016 to 2022 were downloaded and assembled. These were analyzed using the Basic Local Alignment Search Tool (BLAST) with a sequence unique to field strains, followed by a sequence unique to the vaccine strain. The PCR assays were developed against field and vaccine strains by targeting transposition events in the crp and cya genes and validated by screening Salmonella serovar Typhimurium isolates. Between 2016 and 2022, 1708 Salmonella Typhimurium isolates of chicken origin were found in the NCBI Pathogen Detection database, corresponding to 7.99% of all Salmonella identified. Of these, 104 (5.97%) were identified as the vaccine strain. The PCR assay differentiated field strains from the vaccine strain when applied to isolates and was also able to detect the vaccine strain from DNA isolated from mixed serovar overnight Salmonella enrichment cultures. Live attenuated Salmonella vaccines are a critical preharvest tool for Salmonella control and are widely used in industry. With forthcoming regulations that will likely focus on Salmonella Typhimurium, along with other serovars, there is a need to distinguish between isolates belonging to the vaccine strain and those that are responsible for causing human illness.

Impact of water depth on the distributions and proxies of isoprenoidal hydroxylated GDGTs in the Mediterranean Sea and the Red Sea

Hydroxylated Isoprenoidal Glycerol Dialkyl Glycerol Tetraethers (OH-isoGDGTs) have recently been utilized as paleothermometers in the marine environment. However, their ability to reconstruct temperature in the Mediterranean and Red Sea has not been adequately investigated. Previous research has shown that archaeal communities inhabiting different water depths in these basins exert a substantial influence on the regular isoGDGT distributions and associated proxies such as the TEX86. However, the impact of these archaea on OH-isoGDGTs and their corresponding proxies remains unclear. In this study, we examined the distribution of OH-isoGDGTs and their associated proxies (%OH, RI-OH, RI-OH′ and TEX86OH) in surface sediments of the Mediterranean and the Red Sea. We observe strong correlations between the fractional abundances of OH-isoGDGTs, relative to all isoGDGTs and OH-isoGDGTs, and water depth which suggests that deep-water archaeal communities have a lower OH-isoGDGT abundance compared to the shallow communities. As a result, %OH and TEX86OH are strongly correlated with water depth, particularly at depths <500 m in the Mediterranean Sea. Interestingly, RI-OH and RI-OH′ show no correlation with water depth in the Mediterranean Sea. Instead, they correlate more strongly with satellite-derived sea surface temperature compared to other isoGDGT-based proxies, indicating their potential as paleothermometers. Finally, unlike TEX86 and TEX86OH, the %OH, RI-OH and RI-OH′ do not exhibit distinct 'Red Sea cluster' and display comparable values to sediments from other tropical oceans. Further research on sedimentary OH-isoGDGT distributions with broader geographical coverage within these basins and enrichment cultures of deep-water archaea are needed to confirm these observations.

Impact of Organic Carbons Addition on the Enrichment Culture of Nitrifying Biofloc from Aquaculture Water: Process, Efficiency, and Microbial Community.

In this study, we developed a rapid and effective method for enriching the culture of nitrifying bioflocs (NBF) from aquacultural brackish water. The self-designed mixotrophic mediums with a single or mixed addition of sodium acetate, sodium citrate, and sucrose were used to investigate the enrichment process and nitrification efficiency of NBF in small-scale reactors. The results showed that NBF with an MLVSSs from 1170.4 mg L-1 to 2588.0 mg L-1 were successfully enriched in a period of less than 16 days. The citrate group performed the fastest enrichment time of 10 days, while the sucrose group had the highest biomass of 2588.0 ± 384.7 mg L-1. In situ testing showed that the highest nitrification efficiency was achieved in the citrate group, with an ammonia oxidation rate of 1.45 ± 0.34 mg N L-1 h-1, a net nitrification rate of 2.02 ± 0.20 mg N L-1 h-1, and a specific nitrification rate of 0.72 ± 0.14 mg N g-1 h-1. Metagenomic sequencing revealed that Nitrosomonas (0.0~1.0%) and Nitrobacter (10.1~26.5%) were dominant genera for AOB and NOB, respectively, both of which had the highest relative abundances in the citrate group. Linear regression analysis further demonstrated significantly positive linear relations between nitrification efficiencies and nitrifying bacterial genera and gene abundance in NBF. The results of this study provide an efficient enrichment culture method of NBF for the operation of biofloc technology aquaculture systems, which will further promote its wide application in modern intensive aquaculture.

Probing Denitrifying Anaerobic Methane Oxidation via Antimicrobial Intervention: Implications for Innovative Wastewater Management.

Methane emissions present a significant environmental challenge in both natural and engineered aquatic environments. Denitrifying anaerobic methane oxidation (N-DAMO) has the potential for application in wastewater treatment plants. However, our understanding of the N-DAMO process is primarily based on studies conducted on environmental samples or enrichment cultures using metagenomic approaches. To gain deeper insights into N-DAMO, we used antimicrobial compounds to study the function and physiology of 'Candidatus Methanoperedens nitroreducens' and 'Candidatus Methylomirabilis oxyfera' in N-DAMO enrichment cultures. We explored the effects of inhibitors and antibiotics and investigated the potential application of N-DAMO in wastewater contaminated with ammonium and heavy metals. Our results showed that 'Ca. M. nitroreducens' was susceptible to puromycin and 2-bromoethanesulfonate, while the novel methanogen inhibitor 3-nitrooxypropanol had no effect on N-DAMO. Furthermore, 'Ca. M. oxyfera' was shown to be susceptible to the particulate methane monooxygenase inhibitor 1,7-octadiyne and a bacteria-suppressing antibiotic cocktail. The N-DAMO activity was not affected by ammonium concentrations below 10 mM. Finally, the N-DAMO community appeared to be remarkably resistant to lead (Pb) but susceptible to nickel (Ni) and cadmium (Cd). This study provides insights into microbial functions in N-DAMO communities, facilitating further investigation of their application in methanogenic, nitrogen-polluted water systems.

Rebuilding Nursing Culture Through Application of Social Identity Theory and Inclusivity in Health Care Systems: An Exemplar.

This article explores leader practices for rebuilding health system nursing culture by leveraging feedback from clinical nurses and applying Social Identity Theory (SIT) and inclusivity frameworks. An enriched nursing culture is the foundation of quality patient care, and as healthcare systems evolve, it becomes increasingly essential to foster a cohesive and inclusive environment in every aspect of employment practices. Social Identity Theory, which emphasizes how individuals define their self-concept through group affiliations, offers a lens to understand the interplay of identity, values, and behavior within nursing teams. Inclusivity practices are pivotal in creating a welcoming and diverse health care workplace. By employing these approaches, health care systems can rebuild and strengthen their nursing culture, improving retention, onboarding, job satisfaction, teamwork, and enhancing the quality of care provided to patients. This article delves into practical strategies and application of SIT and inclusivity practices to restructure and revitalize nursing culture, emphasizing the positive impact on health care outcomes. An exemplar demonstrating the impact of the voice of the clinician in program development highlights the application of SIT and inclusivity to create culture. It concludes with leader practices for rebuilding nursing culture to include contingent labor as part of the care team.

Phototrophic Nitrogen Fixation, a Neglected Biogeochemical Process in Mine Tailings?

Biological nitrogen fixation (BNF) has important ecological significance in mine tailing by contributing to the initial accumulation of nitrogen. In addition to chemolithotrophic and heterotrophic BNF, light may also fuel BNF in oligotrophic mine tailings. However, knowledge regarding the occurrence and ecological significance of this biogeochemical process in mine tailings remains ambiguous. The current study observed phototrophic BNF in enrichment cultures established from three primary successional stages (i.e., original tailings, biological crusts, and pioneer plants) of tailings. Notably, phototrophic BNF in tailings may be more active at vegetation stages (i.e., biological crusts and pioneering plants) than in bare tailings. DNA-stable isotope probing identified Roseomonas species as potential aerobic anoxygenic phototrophs responsible for phototrophic BNF. Furthermore, metagenomic binning as well as genome mining revealed that Roseomonas spp. contained essential genes involved in nitrogen fixation, anoxygenic photosynthesis, and carbon fixation, suggesting their genetic potential to mediate phototrophic BNF. A causal inference framework equipped with the structural causal model suggested that the enrichment of putative phototrophic diazotrophic Roseomonas may contribute to an elevated total nitrogen content during primary succession in these mine tailings. Collectively, our findings suggest that phototrophic diazotrophs may play important roles in nutrient accumulation and hold the potential to facilitate ecological succession in tailings.

Occurrence of sulfate-reducing bacteria in well water: identification of anaerobic sulfidogenic bacterial enrichment cultures.

Bacteriological studies of well water mainly focus on aerobic and facultative aerobic coliform bacteria. However, the presence of obligate anaerobic bacteria in well water, especially sulfate-reducing bacteria (SRB), possible causative agents of some diseases, is often ignored. In this study, the presence of SRB and coexisting anaerobic bacteria with SRB in sulfate-reducing enrichment cultures obtained from 10 well water samples in Istanbul was investigated. A nested polymerase chain reaction-denaturing gradient gel electrophoresis strategy was performed to characterize the bacterial community structure of the enrichments. The most probable number method was used to determine SRB number. Out of 10, SRB growth was observed in only one (10%) enrichment culture and the SRB number was low (<10 cells/mL). Community members were identified as Desulfolutivibrio sulfodismutans and Anaerosinus sp. The results show that SRB coexist with Anaerosinus sp., and this may indicate poor water quality, posing a risk to public health. Furthermore, Anaerosinus sp., found in the human intestinal tract, may be used as an alternative anaerobic fecal indicator. It is worth noting that the detection of bacteria using molecular analyzes following enrichment culture techniques can bring new perspectives to determine the possible origin and presence of alternative microbial indicators in aquatic environments.

An improved mouse model of sepsis based on intraperitoneal injections of the enriched culture of cecum slurry

AimSepsis is a life-threatening clinical syndrome comprising multiorgan dysfunctions caused by a disproportionate body immune response. There are several animal sepsis models which are based on cecum ligation, cecal puncture, and cecum slurry injection. The major limitation of all current sepsis models is the high variability owing to the variable degree of ligation, puncture and inconsistent microbial composition used for sepsis initiation. The primary objective of this work is to demonstrate the feasibility of a standardized method for sepsis development. Materials and methodsThe cecal slurry bacterial culture was developed and preserved in glycerol stocks. Antibiotics aztreonam and vancomycin were used for generating several defined, enriched cecal slurry bacterial cultures. Mice survival was assessed until 48 hrs post injection, and the tissue samples were collected after 10 hrs from sepsis initiation. Key findingsThe results indicate that increasing polymicrobial load resulted in lower survival rates and was associated with the higher number of infiltrating immune cells and necrosis. H&E (haematoxylin & eosin) staining & serum markers revealed that septic mice exhibited increased inflammation and significant damage to the liver and kidneys. The defined Gram-negative and Gram-positive specific cecal slurry bacterial cultures were developed and their efficiency in inducing sepsis was characterized. SignificanceEnriched cecal slurry bacterial cultures can be stored in glycerol stocks at −80 °C. This has an ethical advantage of avoiding unnecessary animal euthanasia for each experiment and provides a standardization capability of sepsis development.

Bacterial community structure analysis of sludge from Taozi lake and isolation of an efficient 17β-Estradiol (E2) degrading strain Sphingobacterium sp. GEMB-CSS-01

Environmental challenges arising from organic pollutants pose a significant problem for modern societies. Efficient microbial resources for the degradation of these pollutants are highly valuable. In this study, the bacterial community structure of sludge samples from Taozi Lake (polluted by urban sewage) was studied using 16S rRNA sequencing. The bacterial phyla Proteobacteria, Bacteroidetes, and Chloroflexi, which are potentially important in organic matter degradation by previous studies, were identified as the predominant phyla in our samples, with relative abundances of 48.5%, 8.3%, and 6.6%, respectively. Additionally, the FAPROTAX and co-occurrence network analysis suggested that the core microbial populations in the samples may be closely associated with organic matter metabolism. Subsequently, sludge samples from Taozi Lake were subjected to enrichment cultivation to isolate organic pollutant-degrading microorganisms. The strain Sphingobacterium sp. GEMB-CSS-01, tolerant to sulfanilamide, was successfully isolated. Subsequent investigations demonstrated that Sphingobacterium sp. GEMB-CSS-01 efficiently degraded the endocrine-disrupting compound 17β-Estradiol (E2). It achieved degradation efficiencies of 80.0% and 53.5% for E2 concentrations of 10 mg/L and 20 mg/L, respectively, within 10 days. Notably, despite a reduction in degradation efficiency, Sphingobacterium sp. GEMB-CSS-01 retained its ability to degrade E2 even in the presence of sulfanilamide concentrations ranging from 50 to 200 mg/L. The findings of this research identify potential microbial resources for environmental bioremediation, and concurrently provide valuable information about the microbial community structure and patterns within Taozi Lake.

Resuscitation of baijiu pit mud bacteria based on Rpf protein of Umezawaea beigongshangensis.

The pit mud in the Baijiu fermentation cellar is an abundant microbial resource that is closely related to the quality of baijiu. However, many naturally existing species might be in a viable but nonculturable (VBNC) state, posing challenges to the isolation and application of functional species. Herein, a previously isolated strain from baijiu mash, Umezawaea beigongshangensis, was found to contain the rpf gene that encodes resuscitation promotion factor (Rpf). Therefore, the gene was cloned and heterologously expressed, and the recombinant protein (Ub-Rpf 2) was purified. Ub-Rpf 2 was found to significantly promote the growth of resuscitated VBNC state Corynebacterium beijingensis and Sphingomonas beigongshangensis. To determine the resuscitation effect of Ub-Rpf 2 on real ecological samples, the protein was supplemented in pit mud for enrichment culture. Results revealed that specific families and genera were enriched in abundance upon Ub-Rpf 2 incubation, including a new family of Symbiobacteraceae and culturable Symbiobacterium genus. Furthermore, 14 species belonging to 12 genera were obtained in Ub-Rpf 2 treated samples, including a suspected novel species. This study lays a foundation for applying Rpf from U. beigongshangensis to exploit microbial resources in baijiu pit mud.

Development of a closed-tube, calcein-based loop-mediated isothermal amplification assay to detect Salmonella spp. in raw meat samples

Foodborne pathogens compromise food safety and public health, and Salmonella spp. are among the major pathogenic bacteria that cause outbreaks worldwide. Proper surveillance through timely and cost-effective detection methods across the food animal production chain is crucial to prevent Salmonella outbreaks and agricultural losses. Traditional culture methods are labor- and resource-intensive, with lengthy turnaround times. Meanwhile, conventional molecular tools, such as PCR and qPCR, are expensive and require technical skills and equipment. Loop-mediated isothermal amplification (LAMP) is a simple, rapid, inexpensive, highly sensitive, and specific molecular assay that does not require expensive equipment. Hence, this study developed and optimized a closed-tube, calcein-based LAMP assay to detect Salmonella using the invA gene and performed evaluation and validation against conventional PCR. The LAMP assay showed high specificity and sensitivity. It showed 10-fold higher sensitivity than conventional PCR, at <1 ng/μL DNA concentrations. Meanwhile, for CFU/mL, LAMP assay showed 1000-fold higher sensitivity than conventional PCR at 4.8 × 103 cells/mL than 4.8 × 107 cells/mL, respectively. For parallel testing of 341 raw meat samples, after conventional culture enrichment (until Rappaport-Vassiliadis broth), the optimized LAMP assay showed 100% detection on all samples while conventional PCR showed 100%, 99.04%, and 96.64% for raw chicken, beef, and pork samples, respectively. Meanwhile, a shortened enrichment protocol involving 3-h incubation in buffered peptone water only, showed lower accuracy in tandem with the optimized LAMP assay ranging from 55 to 75% positivity rates among samples. These suggest that the optimized LAMP assay possesses higher sensitivity over conventional PCR for invA gene detection when coupled with conventional enrichment culture methods. Hence, this assay has potential as a powerful complementary or alternative Salmonella detection method to increase surveillance capacity and protect consumer food safety and public health worldwide.

Salinity Tolerance and Osmoadaptation Strategies in Four Genera of Anammox Bacteria: Brocadia, Jettenia, Kuenenia, and Scalindua.

The salinity tolerance and osmoadaptation strategies in four phylogenetically distant anammox species, Brocadia, Jettenia, Kuenenia, and Scalindua, were investigated by using highly enriched cell cultures. The first-emerged "Ca. Scalindua sp." showed optimum growth at 1.5-3% salinity and was tolerant to ∼10% salinity (a slight halophile). The second-emerged "Ca. Kuenenia stuttgartiensis" was tolerant to ∼6% salinity with optimum growth at 0.25-1.5% (a halotolerant). These early-emerged "Ca. Scalindua sp." and ″Ca. K. stuttgartiensis" rapidly accumulated K+ ions and simultaneously synthesized glutamate as a counterion. Subsequently, part of the glutamate was replaced by trehalose. In contrast, the late-emerged "Ca. B. sinica" and "Ca. J. caeni" were unable to accumulate sufficient amounts of K+─glutamate and trehalose, resulting in a significant decrease in activity even at 1-2% salinity (nonhalophiles). In addition, the external addition of glutamate may increase anammox activity at high salinity. The species-dependent salinity tolerance and osmoadaptation strategies were consistent with the genetic potential required for the biosynthesis and transport of these osmolytes and the evolutionary history of anammox bacteria: Scalindua first emerged in marine environments and then Kuenenia and other two species gradually expanded their habitat to estuaries, freshwater, and terrestrial environments, while Brocadia and Jettenia likely lost their ability to accumulate K+─glutamate.

Biotransformation of HBCDs by the microbial communities enriched from mangrove sediments

1,2,5,6,9,10-Hexabromocyclododecanes (HBCDs) are a sort of persistent organic pollutants (POPs). This research investigated 12 microbial communities enriched from sediments of four mangroves in China to transform HBCDs. Six microbial communities gained high transformation rates (27.5–97.7%) after 12 generations of serial transfer. Bacteria were the main contributors to transform HBCDs rather than fungi. Analyses on the bacterial compositions and binning genomes showed that Alcanivorax (55.246–84.942%) harboring haloalkane dehalogenase genes dadAH and dadBH dominated the microbial communities with high transformation rates. Moreover, expressions of dadAH and dadBH in the microbial communities and Alcanivorax isolate could be induced by HBCDs. Further, it was found that purified proteins DadAH and DadBH showed high conversion rates on HBCDs in 36 h (91.9 ± 7.4 and 101.0 ± 1.8%, respectively). The engineered Escherichia coli BL21 strains harbored two genes could convert 5.7 ± 0.4 and 35.1 ± 0.1% HBCDs, respectively, lower than their cell-free crude extracts (61.2 ± 5.2 and 56.5 ± 8.7%, respectively). The diastereoisomer-specific transforming trend by both microbial communities and enzymes were γ- > α- > β-HBCD, differed from α- > β- > γ-HBCD by the Alcanivorax isolate. The identified transformation products indicated that HBCDs were dehalogenated via HBr elimination (dehydrobromination), hydrolytic and reductive debromination pathways in the enriched cultures. Two enzymes converted HBCDs via hydrolytic debromination. The present research provided theoretical bases for the biotransformation of HBCDs by microbial community and the bioremediation of HBCDs contamination in the environment.

Enhancing chlamydospore production in Duddingtonia flagrans on solid substrate: The impact of mannitol and varied cultivation conditions

Duddingtonia flagrans is a nematophagous fungus which has shown promising results as a non-chemical parasitic control tool. The fungus disrupts the parasite's life cycle by trapping larvae in the environment through the networks generated from chlamydospores, thus preventing the reinfection of animals. One barrier to the development of a commercial product using this tool is the need to increase chlamydospore production in the laboratory for its administration to livestock. The purpose of this study was to evaluate the addition of mannitol to an enriched culture medium and the effect of adverse cultivation conditions on chlamydospore production. D. flagrans was cultivated on Petri dishes with corn agar for 4 weeks at 27 °C and 70% relative humidity (RH). Four groups were then formed, all with Sabouraud agar as a base, to which different growth inducers were added: GSA (glucose Sabouraud agar), GSA-MI (glucose Sabouraud agar + meso inositol), GSA-E (enriched glucose Sabouraud agar), and AE-M (enriched agar + mannitol). After 4 weeks, chlamydospores were recovered by washing the surface of each plate with distilled water and then quantified. The medium that yielded the highest amount of chlamydospores was subjected to different cultivation conditions: NC (normal conditions): 70% RH and 27 °C, AC (adverse conditions) 1: 20% RH and 40 °C, CA2: 60% RH and 27 °C, and CA3: 55% RH and 24 °C. It was determined that mannitol increases chlamydospore production (65x106 chlamydospores/plate), and when reducing humidity by 10% under cultivation conditions it resulted in an approximately 10% increase in chlamydospore production compared to the control group. These results suggest that the addition of polyols, as well as its cultivation under certain environmental conditions, can improve chlamydospore production on a laboratory scale.

Genomic and functional analysis of the mucinolytic species Clostridium celatum, Clostridium tertium, and Paraclostridium bifermentans.

Mucins are large glycoproteins whose degradation requires the expression of several glycosil hydrolases to catalyze the cleavage of the oligosaccharide chains and release monosaccharides that can be assimilated. In this study, we present a characterization on the strains Clostridium celatum WC0700, Clostridium tertium WC0709, and Paraclostridium bifermentans WC0705. These three strains were previously isolated from enrichment cultures on mucin of fecal samples from healthy subjects and can use mucin as sole carbon and nitrogen source. Genome analysis and in vitro functional analysis of these strains elucidated their physiological and biochemical features. C. celatum WC0700 harbored the highest number of glycosyl hydrolases specific for mucin degradation, while P. bifermentans WC0705 had the least. These predicted differences were confirmed growing the strains on 5 mucin-decorating monosaccharides (L-fucose, N-Acetylneuraminic acid, galactose, N-acetylgalactosamine, and N-acetylglucosamine) as only source of carbon. Fermenting mucin, they all produced formic, acetic, propionic, butyric, isovaleric, and lactic acids, and ethanol; acetic acid was the main primary metabolite. Further catabolic capabilities were investigated, as well as antibiotic susceptibility, biofilm formation, tolerance to oxygen and temperature. The potential pathogenicity of the strains was evaluated through in silico research of virulence factors. The merge between comparative and functional genomics and biochemical/physiological characterization provided a comprehensive view of these mucin degraders, reassuring on the safety of these species and leaving ample scope for deeper investigations on the relationship with the host and for assessing if some relevant health-promoting effect could be ascribed to these SCFA producing species.

Elucidating doxycycline biotransformation mechanism by Chryseobacterium sp. WX1: Multi-omics insights

Doxycycline (DOX) represents a second-generation tetracycline antibiotic that persists as a challenging-to-degrade contaminant in environmental compartments. Despite its ubiquity, scant literature exists on bacteria proficient in DOX degradation. This study marked a substantial advancement in this field by isolating Chryseobacterium sp. WX1 from an activated sludge enrichment culture, showcasing its unprecedented ability to completely degrade 50 mg/L of DOX within 44 h. Throughout the degradation process, seven biotransformation products were identified, revealing a complex pathway that began with the hydroxylation of DOX, followed by a series of transformations. Employing an integrated multi-omics approach alongside in vitro heterologous expression assays, our study distinctly identified the tetX gene as a critical facilitator of DOX hydroxylation. Proteomic analyses further pinpointed the enzymes postulated to mediate the downstream modifications of DOX hydroxylation derivatives. The elucidated degradation pathway encompassed several key biological processes, such as the microbial transmembrane transport of DOX and its intermediates, the orchestration of enzyme synthesis for transformation, energy metabolism, and other gene-regulated biological directives. This study provides the first insight into the adaptive biotransformation strategies of Chryseobacterium under DOX-induced stress, highlighting the potential applications of this strain to augment DOX removal in wastewater treatment systems containing high concentrations of DOX.