Isolation and characterization of a Halomonas species for non-axenic growth-associated production of bio-polyesters from sustainable feedstocks.

Biodegradable plastics are urgently needed to replace petroleum-derived polymeric materials and prevent their accumulation in the environment. To this end, we isolated and characterized a halophilic and alkaliphilic bacterium from the Great Salt Lake in Utah. The isolate was identified as a Halomonas species and designated "CUBES01." Full-genome sequencing and genomic reconstruction revealed the unique genetic traits and metabolic capabilities of the strain, including the common polyhydroxyalkanoate (PHA) biosynthesis pathway. Fluorescence staining identified intracellular polyester granules that accumulated predominantly during the strain's exponential growth, a feature rarely found among natural PHA producers. CUBES01 was found to metabolize a range of renewable carbon feedstocks, including glucosamine and acetyl-glucosamine, as well as sucrose, glucose, fructose, and further glycerol, propionate, and acetate. Depending on the substrate, the strain accumulated up to ~60% of its biomass (dry wt/wt) in poly(3-hydroxybutyrate), while reaching a doubling time of 1.7 h at 30°C and an optimum osmolarity of 1 M sodium chloride and a pH of 8.8. The physiological preferences of the strain may not only enable long-term aseptic cultivation but also facilitate the release of intracellular products through osmolysis. The development of a minimal medium also allowed the estimation of maximum polyhydroxybutyrate production rates, which were projected to exceed 5 g/h. Finally, also, the genetic tractability of the strain was assessed in conjugation experiments: two orthogonal plasmid vectors were stable in the heterologous host, thereby opening the possibility of genetic engineering through the introduction of foreign genes. IMPORTANCE: The urgent need for renewable replacements for synthetic materials may be addressed through microbial biotechnology. To simplify the large-scale implementation of such bio-processes, robust cell factories that can utilize sustainable and widely available feedstocks are pivotal. To this end, non-axenic growth-associated production could reduce operational costs and enhance biomass productivity, thereby improving commercial competitiveness. Another major cost factor is downstream processing, especially in the case of intracellular products, such as bio-polyesters. Simplified cell-lysis strategies could also further improve economic viability.

Bacterial exometabolites influence Chlamydomonas cell cycle and double algal productivity.

Algal-bacterial interactions provide clues to algal physiology, but mutualistic interactions are complicated by dynamic exchange. We characterized the response of Chlamydomonas reinhardtii to the presence of a putative alga-benefitting commensal bacterium (Arthrobacter strain 'P2b'). Co-cultivation promoted chlorophyll content, biomass, average cell size, and number of dividing cells, relative to axenic cultures. Addition of bacterial spent medium (whole, size-fractionated and heat-treated) had similar effects, indicating P2b does not require algal interaction to promote growth. Nutrients and pH were excluded as putative effectors, collectively indicating a commensal interaction mediated by Arthrobacter-released small exometabolite(s). Proteogenomic comparison revealed similar response to co-cultivation and spent media, including differential cell cycle regulation, extensive downregulation of flagellar genes and histones, carbonic anhydrase and RubisCO downregulation, upregulation of some chlorophyll, amino acid and carbohydrate biosynthesis genes, and changes to redox and Fe homeostasis. Further, Arthrobacter protein expression indicated some highly expressed putative secondary metabolites. Together, these results revealed that low molecular weight bacterial metabolites can elicit major physiological changes in algal cell cycle regulation, perhaps through a more productive G1 phase, that lead to substantial increases in photosynthetically-produced biomass. This work illustrates that model commensal interactions can be used to shed light on algal response to stimulating bacteria.

Anaerobic methane oxidation coupled to denitrification is the dominant methane sink in a deep lake.

Anaerobic methane oxidation coupled to denitrification, also known as "nitrate/nitrite-dependent anaerobic methane oxidation" (n-damo), was discovered in 2006. Since then, only a few studies have identified this process and the associated microorganisms in natural environments. In aquatic sediments, the close proximity of oxygen- and nitrate-consumption zones can mask n-damo as aerobic methane oxidation. We therefore investigated the vertical distribution and the abundance of denitrifying methanotrophs related to Candidatus Methylomirabilis oxyfera with cultivation-independent molecular techniques in the sediments of Lake Constance. Additionally, the vertical distribution of methane oxidation and nitrate consumption zones was inferred from high-resolution microsensor profiles in undisturbed sediment cores. M. oxyfera-like bacteria were virtually absent at shallow-water sites (littoral sediment) and were very abundant at deep-water sites (profundal sediment). In profundal sediment, the vertical distribution of M. oxyfera-like bacteria showed a distinct peak in anoxic layers that coincided with the zone of methane oxidation and nitrate consumption, a strong indication for n-damo carried out by M. oxyfera-like bacteria. Both potential n-damo rates calculated from cell densities (660-4,890 µmol CH4⋅m(-2)⋅d(-1)) and actual rates calculated from microsensor profiles (31-437 µmol CH4⋅m(-2)⋅d(-1)) were sufficiently high to prevent methane release from profundal sediment solely by this process. Additionally, when nitrate was added to sediment cores exposed to anoxic conditions, the n-damo zone reestablished well below the sediment surface, completely preventing methane release from the sediment. We conclude that the previously overlooked n-damo process can be the major methane sink in stable freshwater environments if nitrate is available in anoxic zones.