Carmen Pire

Bioplastics such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) are promising alternatives to conventional plastics. However, the high production cost limits their industrial application. In this study, PHBV production was optimized in Haloferax mediterranei by the homologous overexpression of the key enzyme PHA synthase (PhaEC), resulting in the OEphaEC strain. The growth and PHBV production of OEphaEC compared with the parental strain (HM26) were evaluated in three culture media with different nitrogen sources (KNO₃, NH₄Cl, and casamino acids). The OEphaEC strain exhibited a 20% increase in PHBV production and a 40% increase in 3-hydroxyvalerate monomer (3HV) content in a defined medium with nitrate as a nitrogen source, as determined by GC-MS. Moreover, enzyme activity, measured spectrophotometrically, increased from 2.3 to 3.9 U/mg. Soluble and insoluble protein fractions were analysed to assess the overexpression of PHA synthase. Only PhaE was found in the insoluble protein fraction, where PHBV granules accumulate. Transmission electron microscopy (TEM) images confirmed a higher PHBV content in OEphaEC compared to the parental strain. These results demonstrate that the homologous overexpression of the key enzyme implicated in PHBV biosynthesis can enhance PHBV content, making its production competitive for industrial applications.

The haloarchaeon Haloferax mediterranei synthesizes poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) under unfavorable nutritional conditions without the addition of any precursor to the culture, which is an advantage compared to other microbial counterparts able to synthesize polyhydroxyalkanoates (PHA). PHBV is a biodegradable polymer showing physiochemical properties of biotechnological and biomedical interest and can be used as an alternative to plastics made from chemical synthesis (which are not environmentally friendly). The versatile metabolism of H. mediterranei makes the use of waste as a carbon source for cellular growth and PHA synthesis possible. In this work, cellular growth and the production and characterization of PHBV using two different types of confectionery waste were analyzed and compared with cellular growth and PHBV synthesis in a standard culture media with glucose of analytical grade as a carbon source. The PHBV granules produced were analyzed by TEM and the biopolymer was isolated and characterized by GC-MS, FTIR NMR, and DSC. The results reveal that H. mediterranei can use these two residues (R1 and R2) for pure PHBV production, achieving 0.256 and 0.983 g PHBV/L, respectively, which are among the highest yields so far described using for the first-time waste from the candy industry. Thus, a circular economy-based process has been designed to optimize the upscaling of PHBV production by using haloarchaea as cell factories and valorizing confectionery waste.

Denitrification, a crucial biochemical pathway prevalent among haloarchaea in hypersaline ecosystems, has garnered considerable attention in recent years due to its ecological implications. Nevertheless, the underlying molecular mechanisms and genetic regulation governing this respiration/detoxification process in haloarchaea remain largely unexplored. In this study, RNA-sequencing was used to compare the transcriptomes of the haloarchaeon Haloferax mediterranei under oxic and denitrifying conditions, shedding light on the intricate metabolic alterations occurring within the cell, such as the accurate control of the metal homeostasis. Furthermore, the investigation identifies several genes encoding transcriptional regulators and potential accessory proteins with putative roles in denitrification. Among these are bacterioopsin-like transcriptional activators, proteins harboring a domain of unknown function (DUF2249), and cyanoglobin. In addition, the study delves into the genetic regulation of denitrification, finding a regulatory motif within promoter regions that activates numerous denitrification-related genes. This research serves as a starting point for future molecular biology studies in haloarchaea, offering a promising avenue to unravel the intricate mechanisms governing haloarchaeal denitrification, a pathway of paramount ecological importance. IMPORTANCE: Denitrification, a fundamental process within the nitrogen cycle, has been subject to extensive investigation due to its close association with anthropogenic activities, and its contribution to the global warming issue, mainly through the release of N ₂ O emissions. Although our comprehension of denitrification and its implications is generally well established, most studies have been conducted in non-extreme environments with mesophilic microorganisms. Consequently, there is a significant knowledge gap concerning extremophilic denitrifiers, particularly those inhabiting hypersaline environments. The significance of this research was to delve into the process of haloarchaeal denitrification, utilizing the complete denitrifier haloarchaeon Haloferax mediterranei as a model organism. This research led to the analysis of the metabolic state of this microorganism under denitrifying conditions and the identification of regulatory signals and genes encoding proteins potentially involved in this pathway, serving as a valuable resource for future molecular studies.

Halophilic archaea, also termed haloarchaea, are a group of moderate and extreme halophilic microorganisms that constitute the major microbial populations in hypersaline environments. In these ecosystems, mainly aquatic, haloarchaea are constantly exposed to ionic and oxidative stress due to saturated salt concentrations and high incidences of UV radiation (mainly in summer). To survive under these harsh conditions, haloarchaea have developed molecular adaptations including hyperpigmentation. Regarding pigmentation, haloarchaeal species mainly synthesise the rare C₅₀ carotenoid called bacterioruberin (BR) and its derivatives, monoanhydrobacterioruberin and bisanhydrobacterioruberin. Due to their colours and extraordinary antioxidant properties, BR and its derivatives have been the aim of research in several research groups all over the world during the last decade. This review aims to summarise the most relevant characteristics of BR and its derivatives as well as describe their reported antitumoral, immunomodulatory, and antioxidant biological activities. Based on their biological activities, these carotenoids can be considered promising natural biomolecules that could be used as tools to design new strategies and/or pharmaceutical formulas to fight against cancer, promote immunomodulation, or preserve skin health, among other potential uses.

Climate change, global pollution due to plastics, greenhouse gasses, or heavy metals among other pollutants, as well as limited natural sources due to unsustainable lifestyles and consumption patterns, are revealing the need for more research to understand ecosystems, biodiversity, and global concerns from the microscale to the macroscale

Haloferax mediterranei is the model microorganism for the study of the nitrogen cycle in haloarchaea. This archaeon not only assimilate N-species such as nitrate, nitrite, or ammonia, but also it can perform denitrification under low oxygen conditions, using nitrate or nitrite as alternative electron acceptors. However, the information currently available on the regulation of this alternative respiration in this kind of microorganism is scarce. Therefore, in this research, the study of haloarchaeal denitrification using H. mediterranei has been addressed by analyzing the promoter regions of the four main genes of denitrification (narGH, nirK, nor, and nosZ) through bioinformatics, reporter gene assays under oxic and anoxic conditions and by site-directed mutagenesis of the promoter regions. The results have shown that these four promoter regions share a common semi-palindromic motif that plays a role in the control of the expression levels of nor and nosZ (and probably nirK) genes. Regarding the regulation of the genes under study, it has been concluded that nirK, nor, and nosZ genes share some expression patterns, and therefore their transcription could be under the control of the same regulator whereas nar operon expression displays differences, such as the activation by dimethyl sulfoxide with respect to the expression in the absence of an electron acceptor, which is almost null under anoxic conditions. Finally, the study with different electron acceptors demonstrated that this haloarchaea does not need complete anoxia to perform denitrification. Oxygen concentrations around 100 μM trigger the activation of the four promoters. However, a low oxygen concentration per se is not a strong signal to activate the promoters of the main genes involved in this pathway; high activation also requires the presence of nitrate or nitrite as final electron acceptors.

As the association of denitrification with global warming and nitrogen removal from ecosystems has gained attention in recent decades, numerous studies have examined denitrification rates and the distribution of denitrifiers across different environments. In this minireview, reported studies focused on coastal saline environments, including estuaries, mangroves, and hypersaline ecosystems, have been analysed to identify the relationship between denitrification and saline gradients. The analyses of the literature and databases stated the direct effect of salinity on the distribution patterns of denitrifiers. However, few works do not support this hypothesis thus making this topic controversial. The specific mechanisms by which salinity influences denitrifier distribution are not fully understood. Nevertheless, several physical and chemical environmental parameters, in addition to salinity, have been shown to play a role in structuring the denitrifying microbial communities. The prevalence of nirS or nirK denitrifiers in ecosystems is a subject of debate in this work. In general terms, in mesohaline environments, the predominant nitrite reductase is NirS type and, NirK is found predominantly in hypersaline environments. Moreover, the approaches used by different researchers are quite different, resulting in a huge amount of unrelated information, making it difficult to establish comparative analysis. The main techniques used to analyse the distribution of denitrifying populations along salt gradients have been also discussed.