Autotrophic poly-3-hydroxybutyrate accumulation in Cupriavidus necator for sustainable bioplastic production triggered by nutrient starvation.

Cupriavidus necator is a facultative chemolithoautotrophic bacterium able to convert carbon dioxide into poly-3-hydroxybutyrate. This is highly promising as the conversion process allows the production of sustainable and biodegradable plastics. Poly-3-hydroxybutyrate accumulation is known to be induced by nutrient starvation, but information regarding the optimal stress conditions controlling the process is still heterogeneous and fragmentary. This study presents a comprehensive comparison of the effects of nutrient stress conditions, namely nitrogen, hydrogen, phosphorus, oxygen, and magnesium deprivation, on poly-3-hydroxybutyrate accumulation in C. necator DSM545. Nitrogen starvation exhibited the highest poly-3-hydroxybutyrate accumulation, achieving 54% of total cell dry weight after four days of nutrient stress, and a carbon conversion efficiency of 85%. The gas consumption patterns indicated flexible physiological mechanisms underlying polymer accumulation and depolymerization. These findings provide insights into strategies for efficient carbon conversion into bioplastics, and highlight the key role of C. necator for future industrial-scale applications.

Deciphering the genetic landscape of enhanced poly-3-hydroxybutyrate production in Synechocystis sp. B12.

BACKGROUND: Microbial biopolymers such as poly-3-hydroxybutyrate (PHB) are emerging as promising alternatives for sustainable production of biodegradable bioplastics. Their promise is heightened by the potential utilisation of photosynthetic organisms, thus exploiting sunlight and carbon dioxide as source of energy and carbon, respectively. The cyanobacterium Synechocystis sp. B12 is an attractive candidate for its superior ability to accumulate high amounts of PHB as well as for its high-light tolerance, which makes it extremely suitable for large-scale cultivation. Beyond its practical applications, B12 serves as an intriguing model for unravelling the molecular mechanisms behind PHB accumulation. RESULTS: Through a multifaceted approach, integrating physiological, genomic and transcriptomic analyses, this work identified genes involved in the upregulation of chlorophyll biosynthesis and phycobilisome degradation as the possible candidates providing Synechocystis sp. B12 an advantage in growth under high-light conditions. Gene expression differences in pentose phosphate pathway and acetyl-CoA metabolism were instead recognised as mainly responsible for the increased Synechocystis sp. B12 PHB production during nitrogen starvation. In both response to strong illumination and PHB accumulation, Synechocystis sp. B12 showed a metabolic modulation similar but more pronounced than the reference strain, yielding in better performances. CONCLUSIONS: Our findings shed light on the molecular mechanisms of PHB biosynthesis, providing valuable insights for optimising the use of Synechocystis in economically viable and sustainable PHB production. In addition, this work supplies crucial knowledge about the metabolic processes involved in production and accumulation of these molecules, which can be seminal for the application to other microorganisms as well.

A unified compendium of prokaryotic and viral genomes from over 300 anaerobic digestion microbiomes.

BACKGROUND: The anaerobic digestion process degrades organic matter into simpler compounds and occurs in strictly anaerobic and microaerophilic environments. The process is carried out by a diverse community of microorganisms where each species has a unique role and it has relevant biotechnological applications since it is used for biogas production. Some aspects of the microbiome, including its interaction with phages, remains still unclear: a better comprehension of the community composition and role of each species is crucial for a cured understanding of the carbon cycle in anaerobic systems and improving biogas production. RESULTS: The primary objective of this study was to expand our understanding on the anaerobic digestion microbiome by jointly analyzing its prokaryotic and viral components. By integrating 192 additional datasets into a previous metagenomic database, the binning process generated 11,831 metagenome-assembled genomes from 314 metagenome samples published between 2014 and 2022, belonging to 4,568 non-redundant species based on ANI calculation and quality verification. CRISPR analysis on these genomes identified 76 archaeal genomes with active phage interactions. Moreover, single-nucleotide variants further pointed to archaea as the most critical members of the community. Among the MAGs, two methanogenic archaea, Methanothrix sp. 43zhSC_152 and Methanoculleus sp. 52maCN_3230, had the highest number of SNVs, with the latter having almost double the density of most other MAGs. CONCLUSIONS: This study offers a more comprehensive understanding of microbial community structures that thrive at different temperatures. The findings revealed that the fraction of archaeal species characterized at the genome level and reported in public databases is higher than that of bacteria, although still quite limited. The identification of shared spacers between phages and microbes implies a history of phage-bacterial interactions, and specifically lysogenic infections. A significant number of SNVs were identified, primarily comprising synonymous and nonsynonymous variants. Together, the findings indicate that methanogenic archaea are subject to intense selective pressure and suggest that genomic variants play a critical role in the anaerobic digestion process. Overall, this study provides a more balanced and diverse representation of the anaerobic digestion microbiota in terms of geographic location, temperature range and feedstock utilization.

Cupriavidus necator as a platform for polyhydroxyalkanoate production: An overview of strains, metabolism, and modeling approaches.

Cupriavidus necator is a bacterium with a high phenotypic diversity and versatile metabolic capabilities. It has been extensively studied as a model hydrogen oxidizer, as well as a producer of polyhydroxyalkanoates (PHA), plastic-like biopolymers with a high potential to substitute petroleum-based materials. Thanks to its adaptability to diverse metabolic lifestyles and to the ability to accumulate large amounts of PHA, C. necator is employed in many biotechnological processes, with particular focus on PHA production from waste carbon sources. The large availability of genomic information has enabled a characterization of C. necator's metabolism, leading to the establishment of metabolic models which are used to devise and optimize culture conditions and genetic engineering approaches. In this work, the characteristics of available C. necator strains and genomes are reviewed, underlining how a thorough comprehension of the genetic variability of C. necator is lacking and it could be instrumental for wider application of this microorganism. The metabolic paradigms of C. necator and how they are connected to PHA production and accumulation are described, also recapitulating the variety of carbon substrates used for PHA accumulation, highlighting the most promising strategies to increase the yield. Finally, the review describes and critically analyzes currently available genome-scale metabolic models and reduced metabolic network applications commonly employed in the optimization of PHA production. Overall, it appears that the capacity of C. necator of performing CO2 bioconversion to PHA is still underexplored, both in biotechnological applications and in metabolic modeling. However, the accurate characterization of this organism and the efforts in using it for gas fermentation can help tackle this challenging perspective in the future.

Transcriptomic and photosynthetic analyses of Synechocystis sp. PCC6803 and Chlorogloeopsis fritschii sp. PCC6912 exposed to an M-dwarf spectrum under an anoxic atmosphere.

Introduction: Cyanobacteria appeared in the anoxic Archean Earth, evolving for the first time oxygenic photosynthesis and deeply changing the atmosphere by introducing oxygen. Starting possibly from UV-protected environments, characterized by low visible and far-red enriched light spectra, cyanobacteria spread everywhere on Earth thanks to their adaptation capabilities in light harvesting. In the last decade, few cyanobacteria species which can acclimate to far-red light through Far-Red Light Photoacclimation (FaRLiP) have been isolated. FaRLiP cyanobacteria were thus proposed as model organisms to study the origin of oxygenic photosynthesis as well as its possible functionality around stars with high far-red emission, the M-dwarfs. These stars are astrobiological targets, as their longevity could sustain life evolution and they demonstrated to host rocky terrestrial-like exoplanets within their Habitable Zone. Methods: We studied the acclimation responses of the FaRLiP strain Chlorogloeopsis fritschii sp. PCC6912 and the non-FaRLiP strain Synechocystis sp. PCC6803 to the combination of three simulated light spectra (M-dwarf, solar and far-red) and two atmospheric compositions (oxic, anoxic). We first checked their growth, O2 production and pigment composition, then we studied their transcriptional responses by RNA sequencing under each combination of light spectrum and atmosphere conditions. Results and discussion: PCC6803 did not show relevant differences in gene expression when comparing the responses to M-dwarf and solar-simulated lights, while far-red caused a variation in the transcriptional level of many genes. PCC6912 showed, on the contrary, different transcriptional responses to each light condition and activated the FaRLiP response under the M-dwarf simulated light. Surprisingly, the anoxic atmosphere did not impact the transcriptional profile of the 2 strains significantly. Results show that both cyanobacteria seem inherently prepared for anoxia and to harvest the photons emitted by a simulated M-dwarf star, whether they are only visible (PCC6803) or also far-red photons (PCC6912). They also show that visible photons in the simulated M-dwarf are sufficient to keep a similar metabolism with respect to solar-simulated light. Conclusion: Results prove the adaptability of the cyanobacterial metabolism and enhance the plausibility of finding oxygenic biospheres on exoplanets orbiting M-dwarf stars.

Analysis of the anaerobic digestion metagenome under environmental stresses stimulating prophage induction.

BACKGROUND: The viral community has the potential to influence the structure of the microbiome and thus the yield of the anaerobic digestion process. However, the virome composition in anaerobic digestion is still under-investigated. A viral induction experiment was conducted on separate batches undergoing a series of DNA-damaging stresses, in order to coerce temperate viruses to enter the lytic cycle. RESULTS: The sequencing of the metagenome revealed a viral community almost entirely composed of tailed bacteriophages of the order Caudovirales. Following a binning procedure 1,092 viral and 120 prokaryotic genomes were reconstructed, 64 of which included an integrated prophage in their sequence. Clustering of coverage profiles revealed the presence of species, both viral and microbial, sharing similar reactions to shocks. A group of viral genomes, which increase under organic overload and decrease under basic pH, uniquely encode the yopX gene, which is involved in the induction of temperate prophages. Moreover, the in-silico functional analysis revealed an enrichment of sialidases in viral genomes. These genes are associated with tail proteins and, as such, are hypothesised to be involved in the interaction with the host. Archaea registered the most pronounced changes in relation to shocks and featured behaviours not shared with other species. Subsequently, data from 123 different samples of the global anaerobic digestion database was used to determine coverage profiles of host and viral genomes on a broader scale. CONCLUSIONS: Viruses are key components in anaerobic digestion environments, shaping the microbial guilds which drive the methanogenesis process. In turn, environmental conditions are pivotal in shaping the viral community and the rate of induction of temperate viruses. This study provides an initial insight into the complexity of the anaerobic digestion virome and its relation with the microbial community and the diverse environmental parameters. Video Abstract.

Preliminary investigation of microorganisms potentially involved in microplastics degradation using an integrated metagenomic and biochemical approach.

Plastic pollution is becoming an emerging environmental issue due to inappropriate disposal at the end of the materials life cycle. When plastics are released, they undergo physical and chemical corrosion, leading to the formation of small particles, commonly referred to as microplastics. In this study, a microbial community derived from the leachate of a bioreactor containing a mixture of soil and plastic collected during a landfill mining process underwent an enrichment protocol in order to select the microbial species specifically involved in plastic degradation. The procedure was set up and tested on polyethylene, polyvinyl chloride, and polyethylene terephthalate, both in anaerobic and aerobic conditions. The evolution of the microbiome has been monitored using a combined approach based on microscopy, marker-gene amplicon sequencing, genome-centric metagenomics, degradation assays, and GC-MS analyses. This procedure permitted us to deeply investigate the metabolic pathways potentially involved in plastic degradation and to depict the route for microplastics metabolization from the enriched microbial community. Six enzymes, among the ones already identified, were found in our samples (alkane 1-monooxygenase, cutinase, feruloyl esterase, triacylglycerol lipase, medium-chain acyl-CoA dehydrogenase, and protocatechuate 4,5-dioxygenase) and new enzymes, addressed as MHETases most probably for the presence of the catalytic triad (His-Asp-Ser), were detected. Among the enzymes involved in plastics degradation, alkane 1-monooxygenase was found in high copy number (between ten and 62 copies) in the metagenomes that resulted most abundant in the microbiome enriched with polyethylene, while protocatechuate 4,5-dioxygenase was found between one and eight copies in the most abundant metagenomes of the microbial culture enriched with polyethylene terephthalate. Degradation assays, performed using both bacterial lysates and supernatants, revealed interesting results on polyethylene terephthalate degradation. Moreover, this study demonstrates to what extent different types of microplastics can affect the microbial community composition. The results obtained significantly increase the knowledge of the plastic degradation process.

KEMET - A python tool for KEGG Module evaluation and microbial genome annotation expansion.

Background: The rapid accumulation of sequencing data from metagenomic studies is enabling the generation of huge collections of microbial genomes, with new challenges for mapping their functional potential. In particular, metagenome-assembled genomes are typically incomplete and harbor partial gene sequences that can limit their annotation from traditional tools. New scalable solutions are thus needed to facilitate the evaluation of functional potential in microbial genomes. Methods: To resolve annotation gaps in microbial genomes, we developed KEMET, an open-source Python library devised for the analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) functional units. KEMET focuses on the in-depth analysis of metabolic reaction networks to identify missing orthologs through hidden Markov model profiles. Results: We evaluate the potential of KEMET for expanding functional annotations by simulating the effect of assembly issues on real gene sequences and showing that our approach can identify missing KEGG orthologs. Additionally, we show that recovered gene annotations can sensibly increase the quality of draft genome-scale metabolic models obtained from metagenome-assembled genomes, in some cases reaching the accuracy of models generated from complete genomes. Conclusions: KEMET therefore allows expanding genome annotations by targeted searches for orthologous sequences, enabling a better qualitative and quantitative assessment of metabolic capabilities in novel microbial organisms.

Valorization of household food wastes to lactic acid production: A response surface methodology approach to optimize fermentation process.

Lactic acid is a valuable compound used in several industrial processes such as polymers, emulsifiers manufacturing, pharmaceutical, and cosmetic formulations. The present study aims to evaluate the potential use of food waste to produce lactic acid through fermentation, both by indigenous microbiota and by the bio-augmentation with two lactic acid bacteria, namely Lactobacillus plantarum BS17 and Lactobacillus casei BP2. Fermentation was studied both in batch and continuously fed anaerobic reactors at mesophilic conditions and a Response Surface Methodology approach was used to optimize the bioprocess performance and determine the environmental parameters (namely pH and time) that lead to the enhancement of lactic acid production during the batch fermentation by indigenous microorganisms. Results revealed an optimum set of conditions for lactic acid production at a pH value of 6.5 and a fermentation period of 3.5 days at 37 °C. Under these conditions lactic acid production reached a value of 23.07 g/L, which was very similar to the mathematically predicted ones, thus verifying the accuracy of the experimental design. This optimum set of conditions was further employed to examine the production of lactic acid under continuous fermentation operation. Furthermore, concentrations of volatile fatty acids and ethanol were monitored and found to be relatively low, with ethanol being the dominant by-product of fermentation, indicating the presence of heterofermentative bacteria in the food wastes. A final step of downstream process was performed resulting in the successful recovery of lactic acid with purity over 90%.