Applied Microbiology and Biotechnology最新文献

To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies, efficient and scalable expansion procedures must be developed. For other adherent human cell types, the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study, a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings, indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion (({N}_{s1u})), thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics, computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner, improved process understanding allowed an expansion factor of ≈ 26 to be achieved, yielding more than 3 × 10⁹ cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability, identity, and differentiation potential throughout cultivation.

• ({N}_{s1u}) can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors

• Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion

• Perfusion is advantageous and supports the cultivation of hiPSCs

Pollutant-derived risks to human and environmental health are exacerbated by slow natural attenuation rates, often driven by pollutant toxicity to microorganisms that can degrade them or limitations to the ability of microorganisms to metabolise them. This review explores mechanisms employed by bacteria to protect themselves from pollutant toxicity in the context of the evolution of pollutant-degrading abilities. The role of promiscuous enzymes in pollutant transformation is subsequently reviewed, highlighting the emergence of novel metabolic pathways and their transcriptional regulation in response to pollutant exposure, followed by the gene transcription regulation to optimise the cellular component synthesis for adaptation on the novel substrate. Additionally, we discuss epistatic interactions among mutations vital for this process both at macromolecular and at cellular levels. Finally, evolutionary constraints towards enhanced fitness in the context of pollutant degradation are considered, the constraints imposed by the epistasis from mutations on both enzyme level and cellular level, concluding with challenges and emerging opportunities to develop sustainable contaminated site remediation technologies.

•Pollutants can exert toxicity on cellular membrane, enzyme and gene transcription.

•Bacteria can patch promiscuous enzymes into novel pathway to degrade pollutants.

•The evolution trajectory is constrained by epistasis from mutations on enzyme and cellular level.

The evolution of the bacterial community in an up-flow anaerobic reactor with silicone support, continuously fed with pure glycerol (day 0–293) and crude glycerol (day 294–362), was studied. Biomass from a former glycerol-degrading reactor was used as inoculum. The maximum yield and productivity of 1,3-propanediol (PDO) (0.62 mol.mol-gly⁻¹ and 14.7 g.L⁻¹.d⁻¹, respectively) were obtained with crude glycerol. The inoculum had low diversity, with dominance of Lactobacillus (70.6%) and Klebsiella/Raoultella (23.3%). After 293 days of feeding with pure glycerol, the abundance of both taxa decreased to less than 10%, either in the attached biofilm or in the biomass growing in suspension. The genus Clostridium and members of the Ruminococcaceae family then became the majority. In the period after feeding with crude glycerol, Clostridium remained as the majority genus in the biofilm; however, it was partially replaced in the suspension by Eubacterium, a non-glycerol degrading bacterium. This fact, together with the prevalence of other glycerol-degrading genera in the biofilm, such as Caproiciproducens and Lactobacillus, indicated that the bacteria attached to the silicone support were responsible for converting glycerol into 1,3-PDO. Therefore, to increase the 1,3-PDO productivity, a good approach would be to maximize the amount of reactor support. Other genera that do not degrade glycerol, such as Anaerobacter and Acetomaculum, thrived at the expense of cellular decay material. The Canonical Correspondence Analysis demonstrated that the origin of glycerol is an important variable to consider during the bioreactor operation for producing 1,3-PDO, while the glycerol loading rate is not.

• Microbial community showed robustness in a range of operational conditions.

• A significantly high 1,3-propanediol yield can be achieved using crude glycerol.

• The attached biofilm appears to be key to the high production of 1,3-propanediol.

The lignocellulosic biomass (LCB) is an attractive, sustainable, and environmentally friendly alternative to fossil sources to produce biofuel, biomaterials, and biochemicals. However, its recalcitrant and heterogenous structure challenges its biodegradation and valorization. The gut microbiome of soil invertebrate species has emerged as a rich source of LCB-degrading bacteria and enzymes in terrestrial ecosystems. The primary objective of this investigation was to identify the bacterial communities within the Porcellio dilatatus gut (Crustacea: Isopods), to obtain enriched cultures, and to identify bacterial isolates with LCB-degrading activity. A total of 112 enriched cultures were screened, all exhibiting xylanolytic activity. Among them, 94 displayed cellulolytic activity, 30 showed chitinolytic activity, and 21 demonstrated ligninolytic activity. Four enriched cultures were selected, and 128 bacteria with cellulolytic, xylanolytic, chitinolytic, or ligninolytic activity were isolated and taxonomically classified. The obtained results reinforce the potential of bacterial communities within the digestive tract of soil invertebrates as a valuable source of lignocellulose-degrading microorganisms. Thirty-one isolates underwent in-depth enzymatic characterization, and five were selected and functionally evaluated. An artificial bacterial consortium was constructed to assess the potential benefits of using consortia to achieve enhanced LCB degradation. The positive results of this proof-of-concept artificial consortium (PdG-AC) can be used in future applications and is a valuable tool for enzymatic and microbial consortia engineering by, e.g., changing growth conditions for enhanced LCB-degrading abilities.

• The gut microbiome of Porcellio dilatatus was characterized.

• Porcellio dilatatus gut hosts many lignocellulose-degrading bacteria.

• Developed an artificial bacterial consortium for lignocellulose degradation.

One strategy for CO₂ mitigation is using photosynthetic microorganisms to sequester CO₂ under high concentrations, such as in flue gases. While elevated CO₂ levels generally promote growth, excessively high levels inhibit growth through uncertain mechanisms. This study investigated the physiology of the cyanobacterium Synechocystis sp. PCC 6803 under very high CO₂ concentrations and yet stable pH around 7.5. The growth rate of the wild type (WT) at 200 µmol photons m⁻² s⁻¹ and a gas phase containing 30% CO₂ was 2.7-fold lower compared to 4% CO₂. Using a CRISPR interference mutant library, we identified genes that, when repressed, either enhanced or impaired growth under 30% or 4% CO₂. Repression of genes involved in light harvesting (cpc and apc), photochemical electron transfer (cytM, psbJ, and petE), and several genes with little or unknown functions promoted growth under 30% CO₂, while repression of key regulators of photosynthesis (pmgA) and CO₂ capture and fixation (ccmR, cp12, and yfr1) increased growth inhibition under 30% CO₂. Experiments confirmed that WT cells were more susceptible to light inhibition under 30% than under 4% CO₂ and that a light-harvesting-impaired ΔcpcG mutant showed improved growth under 30% CO₂ compared to the WT. These findings suggest that enhanced fitness under very high CO₂ involves modifications in light harvesting, electron transfer, and carbon metabolism, and that the native regulatory machinery is insufficient, and in some cases obstructive, for optimal growth under 30% CO₂. This genetic profiling provides potential targets for engineering cyanobacteria with improved photosynthetic efficiency and stress resilience for biotechnological applications.

• Synechocystis growth was inhibited under very high CO₂.

• Inhibition of growth under very high CO₂ was light dependent.

• Repression of photosynthesis genes improved growth under very high CO₂.

The anaerobic bacterium Clostridium cellulovorans is a promising candidate for the sustainable production of biofuels and platform chemicals due to its cellulolytic properties. However, the genomic engineering of the species is hampered because of its poor genetic accessibility and the lack of genetic tools. To overcome this limitation, a protocol for triparental conjugation was established that enables the reliable transfer of vectors for markerless chromosomal modification into C. cellulovorans. The availability of reporter genes is another requirement for strain engineering and biotechnological applications. In this work, the oxygen-free fluorescence absorption-shift tag (FAST) system was used to characterize promoter strength in C. cellulovorans. Selected promoters were used to establish a CRISPR/Cas system for markerless chromosomal modifications. For stringent control of expression of Cas9, a theophylline-dependent riboswitch was used, and additionally, the anti-CRISPR protein AcrIIA4 was used to reduce the basal activity of the Cas9 in the off-state of the riboswitch. Finally, the newly established CRISPR/Cas system was used for the markerless deletion of the genes encoding two restriction endonucleases of a type II restriction-modification (RS) system from the chromosome of C. cellulovorans. In comparison to the WT, the conjugation efficiency when using the deletion mutant as the recipient strain was improved by about one order of magnitude, without the need for prior C. cellulovorans-specific in vivo methylation of the conjugative plasmid in the E. coli donor strain.

• Quantification of heterologous promoters enables rational choice for genetic engineering.

• CRISPR/Cas with riboswitch and anti-CRISPR allows efficient gene deletion in C. cellulovorans.

• Conjugation protocol and type II REase deletion enhance genetic accessibility.

The rapid advancement of nanotechnology, particularly in the realm of pharmaceutical sciences, has significantly transformed the potential for treating life-threatening diseases. A pivotal aspect of this evolution is the emergence of “green nanotechnology,” which emphasizes the environmentally sustainable synthesis of raw materials through biological processes. This review focuses on the biological synthesis and application of zinc oxide (ZnO) nanoparticles (NPs) from probiotic bacteria, particularly those sourced from wastewater. Microorganisms from wastewater tolerate harmful elements and enzymatically convert toxic heavy metals into eco-friendly materials. These probiotic bacteria are instrumental in the synthesis of ZnO NPs and exhibit remarkable antimicrobial properties with diverse industrial applications. As the challenge of drug-resistant pathogens escalates, innovative strategies for combating microbial infections are essential. This review explores the intersection of nanotechnology, microbiology, and antibacterial resistance, highlighting the importance of selecting suitable probiotic bacteria for synthesizing ZnO NPs with potent antibacterial activity. Additionally, the review addresses the biofunctionalization of NPs and their applications in environmental remediation and therapeutic innovations, including wound healing, antibacterial, and anticancer treatments. Eco-friendly NP synthesis relies on the identification of these suitable microbial “nano-factories.” Targeting probiotic bacteria from wastewater can uncover new microbial NP synthesis capabilities, advancing environmentally friendly NP production methods.

• Innovative strategies are needed to combat drug-resistant pathogens like MRSA.

• Wastewater-derived probiotic bacteria are an eco-friendly method for ZnO synthesis.

• ZnO NPs show significant antimicrobial activity against various pathogens.

The fermentative production of valuable chemicals from lignocellulosic feedstocks has attracted considerable attention. Although Saccharomyces cerevisiae is a promising microbial host, it lacks the ability to efficiently metabolize xylose, a major component of lignocellulosic feedstocks. The xylose oxidative pathway offers advantages such as simplified metabolic regulation and fewer enzymatic steps. Specifically, the pathway involves the conversion of xylose into 2-keto-3-deoxy-xylonate, which can be channeled into two distinct pathways, the Dahms pathway and the Weimberg pathway. However, the growth of yeast on xylose as the sole carbon source through the xylose oxidative pathway has not been achieved, limiting its utilization. We successfully engineered S. cerevisiae to metabolize xylose as its sole carbon source via the xylose oxidative pathways, achieved by enhancing enzyme activities through iron metabolism engineering and rational enzyme selection. We found that increasing the supply of the iron-sulfur cluster to activate the bottleneck enzyme XylD by BOL2 disruption and tTYW1 overexpression facilitated the growth of xylose and the production of ethylene glycol at 1.5 g/L via the Dahms pathway. Furthermore, phylogenetic analysis of xylonate dehydratases led to the identification of a highly active homologous enzyme. A strain possessing the Dahms pathway with this highly active enzyme exhibited reduced xylonate accumulation. Furthermore, the introduction of enzymes based on phylogenetic tree analysis allowed for the utilization of xylose as the sole carbon source through the Weimberg pathway. This study highlights the potential of iron metabolism engineering and phylogenetic enzyme selection for the development of non-native metabolic pathways in yeast.

• A 1.5 g/L ethylene glycol was produced via the Dahms pathway in S. cerevisiae.

• Enzyme activation enabled growth on xylose via both the Dahms and Weimberg pathways.

• Tested enzymes in this study may expand the application of xylose oxidative pathway.

Microbial fuel cell (MFC) technology has received increased interest as a suitable approach for treating wastewater while producing electricity. However, there remains a lack of studies investigating the impact of inoculum type and hydraulic retention time (HRT) on the efficiency of MFCs in treating industrial saline wastewater. The effect of three different inocula (activated sludge from a fish-canning industry and two domestic wastewater treatment plants, WWTPs) on electrochemical and physicochemical parameters and the anodic microbiome of a two-chambered continuous-flow MFC was studied. For each inoculum, three different HRTs were tested (1 day, 3 days, and 6 days). The inoculum from the fish canning industry significantly increased voltage production (with a maximum value of 802 mV), power density (with a maximum value of 78 mW m⁻²), coulombic efficiency (with a maximum value of 19.3%), and organic removal rate (ORR) compared to the inocula from domestic WWTPs. This effect was linked to greater absolute and relative abundances of electroactive microorganisms (e.g., Geobacter, Desulfovibrio, and Rhodobacter) and predicted electron transfer genes in the anode microbiome likely due to better adaption to salinity conditions. The ORR and current production were also enhanced at shorter HRTs (1 day vs. 3 and 6 days) across all inocula. This effect was related to a greater abundance and diversity of bacterial communities at HRT of 1 day compared to longer HRTs. Our findings have important bioengineering implications and can help improve the performance of MFCs treating saline effluents such as those from the seafood industry.

• Inoculum type and HRT impact organic matter removal and current production.

• Changes in bioenergy generation were linked to the electroactive anodic microbiome.

• Shorter HRT favored increases in the performance of the MFC.