Quorum quenching of autoinducer 2 increases methane production in anaerobic digestion of waste activated sludge.

The ubiquitous signaling molecule autoinducer 2 (AI-2) is involved in intra- and interspecies communication, most notably between Gram-negative and Gram-positive bacteria. AI-2 accumulates during the exponential phase of the Escherichia coli (E. coli) monoculture and then rapidly decreases upon entry into the stationary phase. However, deleting both the genes encoding AI-2 synthase (LuxS) and the lsr operon regulator (LsrR) in the E. coli genome causes impaired AI-2 production and continuous AI-2 scavenging from the environment. This genetically-engineered E. coli mutant capable of quenching AI-2 quorum sensing (QS) system was utilized to evaluate the effect of AI-2 quenching on the anaerobic digestion of waste activated sludge (WAS) because the role of QS system via AI-2 in the process remains obscure. In this study, E. coli ∆luxS lsrR mutant cells were microencapsulated in sodium alginate beads and incubated with WAS anaerobically. After 15 days of anaerobic fermentation, the WAS containing double mutant cells produced significantly more methane than that of the parent E. coli cells. AI-2 quenching occurred concurrently with a shift of microbial communities that contribute to increasing acetate consumption by the Methanosarcina spp. resulting in an increase in methane production. KEY POINTS: • Impact of autoinducer 2 quenching in complex bacterial populations were determined. • Key microorganisms contributing to the increase of methane in WAS anaerobic digestion were found. • The AI-2 quenching is a potential regulatory in wastewater treatment and bioenergy research.

Engineering anaerobic digestion via optimizing microbial community: effects of bactericidal agents, quorum sensing inhibitors, and inorganic materials.

Anaerobic digestion of sewage sludge (SS) is one of the effective ways to reduce the waste generated from human life activities. To date, there are many reports to improve or repress methane production during the anaerobic digestion of SS. In the anaerobic digestion process, many microorganisms work positively or negatively, and as a result of their microbe-to-microbe interaction and regulation, methane production increases or decreases. In other words, understanding the complex control mechanism among the microorganisms and identifying the strains that are key to increase or decrease methane production are important for promoting the advanced production of bioenergy and beneficial compounds. In this mini-review, the literature on methane production in anaerobic digestion has been summarized based on the results of antibiotic addition, quorum sensing control, and inorganic substance addition. By optimizing the activity of microbial groups in SS, methane or acetate can be highly produced. KEY POINTS: • Bactericidal agents such as an antibiotic alter microbial community for enhanced CH4 production. • Bacterial interaction via quorum sensing is one of the key points for biofilm and methane production. • Anaerobic digestion can be altered in the presence of several inorganic materials.

Diluted Luria-Bertani medium vs. sewage sludge as growth media: comparison of community structure and diversity in the culturable bacteria.

Because colony formation is essential to seek bacterial functions by the direct observation of phenotype, the diversification of colony formation for culturable bacteria is a big challenge in the research field of Environmental Biotechnology. In this study, the biodiversity of cultivable bacteria (colony or liquid culture) was compared by using Luria-Bertani (LB) medium and waste sewage sludge (WSS) under different dilutions and temperatures. When WSS was used as a bacterial source, whereas the highest number of colonies was found at the concentration of WSS (5%), a particular concentration of LB (10%) or WSS (1%) as a growth medium showed the best number of the operational taxonomic units (OTUs) of colonies. The results of bacterial community structure indicated that there are 1, 8, and 12 bacterial genera found uniquely in the agar plates of LB, 10% LB, and 5% WSS. By contrast, when palm oil mill effluent sludge was used as a bacterial source, the effect of dilution was different with WSS. When comparing the biodiversity between colonies and liquid culture, a high OTU value was observed in the colonies on the plate. In addition, 30°C showed the highest number of colonies in LB, 10% LB, and 5% WSS whereas the best OTUs were observed at 37°C for LB and 10% LB, and at 25°C for 5% WSS. This study demonstrates the diversification of cultivable bacteria through the number of OTUs in diluted LB medium and WSS, which is beneficial to isolate a unique bacterial strain.Key points• Impacts of diluted LB medium and WSS for colony formation were determined.• Difference of concentration of LB and WSS made different effects on colony formation.• Temperature change affected on diluted LB and WSS as media.

Recovery of recombinant Mycobacterium tuberculosis antigens fused with cell wall-anchoring motif (LysM) from inclusion bodies using non-denaturing reagent (N-laurylsarcosine).

BACKGROUND: The current limitations of conventional BCG vaccines highlights the importance in developing novel and effective vaccines against tuberculosis (TB). The utilization of probiotics such as Lactobacillus plantarum for the delivery of TB antigens through in-trans surface display provides an effective and safe vaccine approach against TB. Such non-recombinant probiotic surface display strategy involves the fusion of candidate proteins with cell wall binding domain such as LysM, which enables the fusion protein to anchor the L. plantarum cell wall externally, without the need for vector genetic modification. This approach requires sufficient production of these recombinant fusion proteins in cell factory such as Escherichia coli which has been shown to be effective in heterologous protein production for decades. However, overexpression in E. coli expression system resulted in limited amount of soluble heterologous TB-LysM fusion protein, since most of it are accumulated as insoluble aggregates in inclusion bodies (IBs). Conventional methods of denaturation and renaturation for solubilizing IBs are costly, time-consuming and tedious. Thus, in this study, an alternative method for TB antigen-LysM protein solubilization from IBs based on the use of non-denaturating reagent N-lauroylsarcosine (NLS) was investigated. RESULTS: Expression of TB antigen-LysM fusion genes was conducted in Escherichia coli, but this resulted in IBs deposition in contrast to the expression of TB antigens only. This suggested that LysM fusion significantly altered solubility of the TB antigens produced in E. coli. The non-denaturing NLS technique was used and optimized to successfully solubilize and purify ~ 55% of the recombinant cell wall-anchoring TB antigen from the IBs. Functionality of the recovered protein was analyzed via immunofluorescence microscopy and whole cell ELISA which showed successful and stable cell wall binding to L. plantarum (up to 5 days). CONCLUSION: The presented NLS purification strategy enables an efficient and rapid method for obtaining higher yields of soluble cell wall-anchoring Mycobacterium tuberculosis antigens-LysM fusion proteins from IBs in E. coli.

Proof of concept in utilizing in-trans surface display system of Lactobacillus plantarum as mucosal tuberculosis vaccine via oral administration in mice.

BACKGROUND: Tuberculosis is one of the most common and deadliest infectious diseases worldwide affecting almost a third of the world's population. Although this disease is being prevented and controlled by the Bacille Calmette Guérin (BCG) vaccine, the protective efficacy is highly variable and substandard (0-80%) in adults. Therefore, novel and effective tuberculosis vaccine that can overcome the limitations from BCG vaccine need to be developed. RESULTS: A novel approach of utilizing an in-trans protein surface display system of Lactobacillus plantarum carrying and displaying combination of Mycobacterium tuberculosis subunit epitope antigens (Ag85B, CFP-10, ESAT-6, Rv0475 and Rv2031c) fused with LysM anchor motif designated as ACERL was constructed, cloned and expressed in Esherichia coli Rossetta expression host. Subsequently the binding capability of ACERL to the cell wall of L. plantarum was examined via the immunofluorescence microscopy and whole cell ELISA where successful attachment and consistent stability of cell wall binding up to 4 days was determined. The immunization of the developed vaccine of L. plantarum surface displaying ACERL (Lp ACERL) via the oral route was studied in mice for its immunogenicity effects. Lp ACERL immunization was able to invoke significant immune responses that favor the Th1 type cytokine response of IFN-γ, IL-12 and IL-2 as indicated by the outcome from the cytokine profiling of spleen, lung, gastrointestinal tract (GIT), and the re-stimulation of the splenocytes from the immunized mice. Co-administration of an adjuvant consisting of Lactococcus lactis secreting mouse IL-12 (LcIL-12) with Lp ACERL was also investigated. It was shown that the addition of LcIL-12 was able to further generate significant Th1 type cytokines immune responses, similar or better than that of Lp ACERL alone which can be observed from the cytokine profiling of the immunized mice's spleen, lung and GIT. CONCLUSIONS: This study represents a proof of concept in the development of L. plantarum as a carrier for a non-genetically modified organism (GMO) tuberculosis vaccine, which may be the strategy in the future for tuberculosis vaccine development.

Cu2O/TiO2 decorated on cellulose nanofiber/reduced graphene hydrogel for enhanced photocatalytic activity and its antibacterial applications.

Photocatalysis has gained attention as a viable wastewater remediation technique. However, the difficulty of recovering powder-based photocatalyst has often become a major limitation for their on-site practical application. Herein, we report on the successful in-situ preparation of a novel three-dimensional (3D) photocatalyst consisting of Cu2O/TiO2 loaded on a cellulose nanofiber (CNF)/reduced graphene hydrogel (rGH) via facile hydrothermal treatment and freeze-drying. The 3D macrostructure not only provides a template for the anchoring of Cu2O and TiO2 but also provides an efficient electron transport pathway for enhanced photocatalytic activity. The results showed that the Cu2O and TiO2 were uniformly loaded onto the aerogel framework resulting in the composites with large surface area with exposed actives sites. As compared to bare rGH, CNF/rGH, Cu2O/CNF/rGH and TiO2/CNF/rGH, the Cu2O/TiO2/CNF/rGH showed improved photocatalytic activity for methyl orange (MO) degradation. MO degradation pathway is proposed based on GC-MS analysis. The enhanced photoactivity can be attributed to the charge transfer and electron-hole separation from the synergistic effect of Cu2O/TiO2 anchored on CNF/rGH. In terms of their anti-bacterial activity towards Staphylococcus aureus and Escherichia coli, the synergistic effect of the Cu2O/TiO2 anchored on the CNF/rGH framework showed excellent activity towards the bacteria.

Safety assessment of fermented jackfruit (Artocarpus heterophyllus) pulp and leaves in Sprague-Dawley rats.

Fermented jackfruit (Artocarpus heterophyllus) extracts were produced using pure symbiotic culture of bacteria and yeast (SCOBY) under controlled fermentation process. Both female and male Sprague-Dawley rats were orally administrated with 4,000 mg/kg of fermented jackfruit pulp and leaves extracts for 28 consecutive days. Body weight of rats was recorded at 1-week interval until necropsy day. There was no mortality reported along the experiment with no significant differences (p > .05) record among organ histopathology and blood biochemical parameters in treated groups when compared to control group. Interestingly, there were significant differences (p < .05) in the lower body weight gained of treated rats groups as opposed to control group, indicating the potential anti-obesity effect of fermented jackfruit extracts. In conclusion, no toxicity symptoms were observed in 28 days oral administration toxicity study of fermented jackfruit pulp and leaves extracts in Sprague-Dawley rats for both sexes.