Halophilic archaea as tools for bioremediation technologies.

Haloarchaea are extremophilic microorganisms belonging to the Archaea domain that require high salt concentrations to be alive, thus inhabiting ecosystems like salty ponds, salty marshes, or extremely salty lagoons. They are more abundantly and widely distributed worldwide than initially expected. Most of them are grouped into two families: Halobacteriaceae and Haloferacaceae. The extreme conditions under which haloarchaea survive contribute to their metabolic and molecular adaptations, thus making them good candidates for the design of bioremediation strategies to treat brines, salty water, and saline soils contaminated with toxic compounds such as nitrate, nitrite, oxychlorates such as perchlorate and chlorate, heavy metals, hydrocarbons, and aromatic compounds. New advances in understanding haloarchaea physiology, metabolism, biochemistry, and molecular biology suggest that biochemical pathways related to nitrogen and carbon, metals, hydrocarbons, or aromatic compounds can be used for bioremediation proposals. This review analyses the novelty of the most recent results showing the capability of some haloarchaeal species to assimilate, modify, or degrade toxic compounds for most living beings. Several examples of the role of these microorganisms in the treatment of polluted brine or salty soils are also discussed in connection with circular economy-based processes. KEY POINTS: • Haloarchaea are extremophilic microorganisms showing genuine metabolism • Haloarchaea can metabolise compounds that are highly toxic to most living beings • These metabolic capabilities are useful for designing soil and water bioremediation strategies.

Transcriptomic profiling of haloarchaeal denitrification through RNA-Seq analysis.

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.IMPORTANCEDenitrification, 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 N2O 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.

Bacterioruberin: Biosynthesis, Antioxidant Activity, and Therapeutic Applications in Cancer and Immune Pathologies.

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.

Molecular Advances in Microbial Metabolism 2.0.

The advances in molecular biology techniques and omics approaches have made it possible to take giant steps in applied research in life sciences [...].

Denitrification in hypersaline and coastal environments.

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.

Haloarchaeal carotenoids exert an in vitro antiproliferative effect on human breast cancer cell lines.

Oxidative stress has been linked to the onset and progression of different neoplasia. Antioxidants might help prevent it by modulating biochemical processes involved in cell proliferation. Here, the aim was to evaluate the in vitro cytotoxic effect of Haloferax mediterranei bacterioruberin-rich carotenoid extracts (BRCE) (0-100 µg/ml) in six BC cell lines, representative of the intrinsic phenotypes and a healthy mammary epithelium cell line. Cell index values were obtained using xCELLigence RTCA System. Furthermore, cell diameter, viability, and concentration were measured at 12 h, 24 h, and 30 h. We found that BC cells were selectively affected by BRCE (SI > 1, p < 0.005). After 30 h, the population of BC cells exposed to 100 µg/ml was 11.7-64.6% of the control (p = 0.0001-0.0009). Triple-negative cells were significantly affected [MDA-MB-231 (IC50 51.8 µg/ml, p < 0.0001) and MDA-MB-468 (IC50 63.9 µg/ml, p < 0.0001)]. Cell size was also reduced after 30 h treatment in 3.8 (± 0.1) µm and 3.3 (± 0.02) µm for SK-BR-3 (p < 0.0001) and MDA-MB-468 (p < 0.0001), respectively. In conclusion, Hfx. mediterranei BRCE exerts a cytotoxic effect on BC cell lines representative of all studied intrinsic subtypes. Furthermore, results obtained for MDA-MB-231 and MDA-MB-468 are very promising, considering the aggressive behaviour of the triple-negative BC subtype.

Molecular Advances in Microbial Metabolism.

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 [...].

Bioactive molecules from haloarchaea: Scope and prospects for industrial and therapeutic applications.

Marine environments and salty inland ecosystems encompass various environmental conditions, such as extremes of temperature, salinity, pH, pressure, altitude, dry conditions, and nutrient scarcity. The extremely halophilic archaea (also called haloarchaea) are a group of microorganisms requiring high salt concentrations (2-6 M NaCl) for optimal growth. Haloarchaea have different metabolic adaptations to withstand these extreme conditions. Among the adaptations, several vesicles, granules, primary and secondary metabolites are produced that are highly significant in biotechnology, such as carotenoids, halocins, enzymes, and granules of polyhydroxyalkanoates (PHAs). Among halophilic enzymes, reductases play a significant role in the textile industry and the degradation of hydrocarbon compounds. Enzymes like dehydrogenases, glycosyl hydrolases, lipases, esterases, and proteases can also be used in several industrial procedures. More recently, several studies stated that carotenoids, gas vacuoles, and liposomes produced by haloarchaea have specific applications in medicine and pharmacy. Additionally, the production of biodegradable and biocompatible polymers by haloarchaea to store carbon makes them potent candidates to be used as cell factories in the industrial production of bioplastics. Furthermore, some haloarchaeal species can synthesize nanoparticles during heavy metal detoxification, thus shedding light on a new approach to producing nanoparticles on a large scale. Recent studies also highlight that exopolysaccharides from haloarchaea can bind the SARS-CoV-2 spike protein. This review explores the potential of haloarchaea in the industry and biotechnology as cellular factories to upscale the production of diverse bioactive compounds.

Analysis of the external signals driving the transcriptional regulation of the main genes involved in denitrification in Haloferax mediterranei.

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.

Evaluation of Haloferax mediterranei Strain R4 Capabilities for Cadmium Removal from Brines.

Haloferax mediterranei has revealed a high bioremediation potential for several inorganic anions (e.g., nitrates and nitrites) and metals from hypersaline waters and brines. However, it is unclear, to date, whether this microorganism allows Cd (II) bioremediation. Consequently, the main objective of this work was to assess the Cd (II) bioremediation potential of Hfx. mediterranei R4. To this end, Hfx. mediterranei cell growth rate and metal bioaccumulation were investigated using different culture media (complex, CM, and defined medium, DM) containing Cd (II) up to 1 mM. In addition, the elemental profile of the biomass (i.e., Al, Ba, Ca, Co, Cu, Fe, K, Mg, Mn, Na, Ni, Sr and Zn) has also been monitored to gain insight into the metabolic processes that may be taking place at the intracellular level for Cd (II) removal. Because of the formation of CdS precipitate, CM is not a suitable culture media for evaluating Cd bioremediation since metal concentration could not be appropriately controlled. When operating in DM, it was observed that the cell doubling time increases three times in the presence of Cd (II). Hfx. mediterranei can bioaccumulate Cd, showing the highest significant accumulation at concentrations of 0.4 mM (108 ± 12 mg Cd/g dry tissue). Finally, the presence of Cd (II) affects the content of K, Mg, Mn and Zn in the biomass, by increasing K levels up to 27 ± 18% and Mn up to 310 ± 140% and reducing Mg levels up to 55 ± 36% and Zn up to 30 ± 4%. These results suggest that different mechanisms are involved in Cd (II) tolerance by Hfx. mediterranei, resulting in increasing the cell concentration of stress-tolerant elements in the biomass (K and Mn), while lowering the concentration of elements which Cd (II) competes with (Mg and Zn), and that all affects the physiological response of the organism by decreasing its growth rate.

Carbon Source Influences Antioxidant, Antiglycemic, and Antilipidemic Activities of Haloferax mediterranei Carotenoid Extracts.

Haloarchaeal carotenoids have attracted attention lately due to their potential antioxidant activity. This work studies the effect of different concentrations of carbon sources on cell growth and carotenoid production. Carotenoid extract composition was characterized by HPLC-MS. Antioxidant activity of carotenoid extracts obtained from cell cultures grown under different nutritional conditions was determined by 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH), Ferric Reducing Ability Power (FRAP) and ß-carotene bleaching assays. The ability of these carotenoid extracts to inhibit α-glucosidase, α-amylase, and lipase enzymes was also assessed to determine if they could be used to reduce blood glucose and lipid absorption. The maximum production of carotenoids (92.2 µg/mL) was observed combining 12.5% inorganic salts and 2.5% of glucose/starch. Antioxidant, hypoglycemic, and antilipidemic studies showed that higher carbon availability in the culture media leads to changes in the extract composition, resulting in more active haloarchaeal carotenoid extracts. Carotenoid extracts obtained from high-carbon-availability cell cultures presented higher proportions of all-trans-bacterioruberin, 5-cis-bacterioruberin, and a double isomeric bacterioruberin, whereas the presence 9-cis-bacterioruberin and 13-cis-bacterioruberin decreased. The production of haloarchaeal carotenoids can be successfully optimized by changing nutritional conditions. Furthermore, carotenoid composition can be altered by modifying carbon source concentration. These natural compounds are very promising in food and nutraceutical industries.

Characterization of Polyhydroxybutyrate, PHB, Synthesized by Newly Isolated Haloarchaea Halolamina spp.

This work aims to characterize the haloarchaeal diversity of unexplored environmental salty samples from a hypersaline environment on the southern coast of Jeddah, Saudi Arabia, looking for new isolates able to produce polyhydroxyalkanoates (PHAs). Thus, the list of PHA producers has been extended by describing two species of Halolamina; Halolamina sediminis sp. strain NRS_35 and unclassified Halolamina sp. strain NRS_38. The growth and PHA-production were investigated in the presence of different carbon sources, (glucose, sucrose, starch, carboxymethyl cellulose (CMC), and glycerol), pH values, (5-9), temperature ranges (4-65 °C), and NaCl concentrations (100-350 g L-1). Fourier-transform infra-red analysis (FT-IR) and Liquid chromatography-mass spectrometry (LC-MS) were used for qualitative identification of the biopolymer. The highest yield of PHB was 33.4% and 27.29% by NRS_35 and NRS_38, respectively, using starch as a carbon source at 37 °C, pH 7, and 25% NaCl (w/v). The FT-IR pattern indicated sharp peaks formed around 1628.98 and 1629.28 cm-1, which confirmed the presence of the carbonyl group (C=O) on amides and related to proteins, which is typical of PHB. LC-MS/MS analysis displayed peaks at retention times of 5.2, 7.3, and 8.1. This peak range indicates the occurrence of PHB and its synthetic products: Acetoacetyl-CoA and PHB synthase (PhaC). In summary, the two newly isolated Halolamina species showed a high capacity to produce PHB using different sources of carbon. Further research using other low-cost feedstocks is needed to improve both the quality and quantity of PHB production. With these results, the use of haloarchaea as cell factories to produce PHAs is reinforced, and light is shed on the global concern about replacing plastics with biodegradable polymers.

Hypersaline environments as natural sources of microbes with potential applications in biotechnology: The case of solar evaporation systems to produce salt in Alicante County (Spain).

Extremophilic microbes show a unique metabolism due to the adaptations they display to deal with extreme environmental parameters characterizing the extreme ecosystems that they inhabit (high salt concentration, high temperatures, and extreme pH values, high exposure to solar radiation etc.). Halophilic microorganisms characterised and isolated from saltmarshes, brines, salted ponds, salty lagoons etc. have recently attracted attention due to their potential biotechnological applications (as whole cells used for different purposes like wastewater treatments, or their biomolecules: enzymes, antibiotics, carotenoids, bioplastics). Alicante county (southeast of Spain) accounts for a significant number of salty environments like coastal or inland salty ponds from where sodium chloride (NaCl)is obtained, marshes, salty lagoons, etc. The best system characterised so far from a microbiological point of view is "Salinas de Santa Pola", also termed "Salinas Bras del Port". However, there are many other salty environments to be explored, like the natural park of Torrevieja and la Mata lagoons, salty lagoon located in Calpe city or inland salted ponds like those located in the northwest of the county. This review summarises the most relevant biotechnological applications of halophilic microbes described up to now. In addition, special attention is focused on ecosystems such as the lagoons of Torrevieja or inland salt marshes as natural environments whose microbial biodiversity is worthy of being studied in search of new strains and species with the aim to analyze their potential biotechnological applications (pharmaceutical, food industry, biomedicine, etc.).

Assessment of Haloferax mediterranei Genome in Search of Copper-Molecular Machinery With Potential Applications for Bioremediation.

Heavy metals are essential micronutrients at low concentrations, serving as cofactors for relevant microbial enzymes (i.e., respiratory nitrate and nitrite reductases NADH dehydrogenase-2, amine oxidase, etc.), but they become harmful cellular intoxicants at significant low concentrations compared to other chemical compounds. The increasing need to incorporate bioremediation in the removal of heavy metals and other contaminants from wastewaters has led extremophiles to the spotlight of research. The haloarchaeon Haloferax mediterranei has promising physiological characteristics regarding bioremediation. However, little is known about how haloarchaea manage to resist high concentrations of heavy metals in the environment. The aim of this work is to develop bioinformatics research as the first step for further omics-based studies to shed light on copper metabolism in haloarchaea by analyzing H. mediterranei genome (strain ATCC 33500). To reach this aim, genome and protein databases have been consulted, and copper-related genes have been identified. BLAST analysis has been carried out to find similarities between copper resistance genes described from other microorganisms and H. mediterranei genes. Plausible copper importer genes, genes coding for siderophores, and copper exporters belonging to P1B-type ATPase group have been found apart from genes encoding copper chaperones, metal-responsive transcriptional regulators, and several proteins belonging to the cupredoxin superfamily: nitrite reductase, nitrous oxide reductases, cytochrome c oxidases, multicopper oxidases, and small blue copper proteins from the amicyanin/pseudoazurin families as halocyanins. As the presence of heavy metals causes oxidative stress, genes coding for proteins involved in antioxidant mechanisms have been also explored: thioredoxin, glutaredoxin, peroxiredoxin, catalase, and γ-glutamylcysteine as an analog of glutathione. Bioinformatic-based analysis of H. mediterranei genome has revealed a set of genes involved in copper metabolism that could be of interest for bioremediation purposes. The analysis of genes involved in antioxidative mechanisms against heavy metals makes it possible to infer the capability of H. mediterranei to synthesize inorganic polyphosphate granules against oxidative stress.

Biosynthesis of Silver Chloride Nanoparticles (AgCl-NPs) from Extreme Halophiles and Evaluation of Their Biological Applications.

The biosynthesis of nanoparticles (NPs) has gained an overwhelming interest due to their biological applications. However, NPs synthesis by pigmented extreme halophiles remains underexplored. The NPs synthesis using pigmented halophiles is inexpensive and less toxic than other processes. In this study, pigmented halophilic microorganisms (n = 77) were screened to synthesize silver chloride nanoparticles (AgCl-NPs) with silver nitrate as metal precursors, and their biological applications were assessed. The synthesis of AgCl-NPs was possible using the crude extract from cellular lysis (CECL) of six extreme halophiles. Two of the AgCl-NPs viz. AK2-NPs and MY6-NPs synthesized by the CECL of Haloferax alexandrinus RK_AK2 and Haloferax lucentense RK_MY6, respectively, exhibited antimicrobial, antioxidative, and anti-inflammatory activities. The surface plasmon resonance of the AgCl-NPs was determined with UV spectroscopy. XRD analysis of AK2-NPs and MY6-NPs confirmed the presence of silver in the form of chlorargyrite (silver chloride) having a cubic structure. The crystallite size of AK2-NPs and MY6-NPs, estimated with the Scherrer formula, was 115.81 nm and 137.50 nm. FTIR analysis verified the presence of diverse functional groups. Dynamic light-scattering analysis confirmed that the average size distribution of NPs was 71.02 nm and 117.36 nm for AK2-NPs and MY6-NPs, respectively, with monodisperse nature. The functional group in 1623-1641 cm-1 indicated the presence of protein ß-sheet structure and shifting of amino and hydroxyl groups from the pigmented CECL, which helps in capping and stabilizing nanoparticles. The study provides evidence that CECL of Haloferax species can rapidly synthesize NPs with unique characteristics and biological applications.

Controversy on the toxic nature of deep eutectic solvents and their potential contribution to environmental pollution.

Deep eutectic solvents (DES) are promising reaction media where interesting catalytic processes can be carried out. In theory, most of these mixtures are environmentally friendly, being an alternative to traditionally pollutant organic solvents used in several processes related to organic chemistry and biotechnology. However, recent studies show contradictory results regarding their toxicity. The method selected to perform toxicity studies could be significantly conditioned by some of the physical properties displayed by the DESs. Also, the metabolic capabilities of the organisms/cells used to monitor their toxicity are influenced by their physical properties. In this review, relevant physical-chemical properties for toxicity studies are summarized. The advantages/disadvantages of the used tests to monitor their toxicity and biodegradability in connection with the chosen organisms/cells are discussed, shedding light on their limitations. These findings could be taken as a starting point for designing more accurate DESs toxicity studies covering a wider spectrum of organisms and cells to be used as biomodels to monitor environmental pollution caused by DESs.

Halophilic Carotenoids and Breast Cancer: From Salt Marshes to Biomedicine.

Breast cancer is the leading cause of death among women worldwide. Over the years, oxidative stress has been linked to the onset and progression of cancer. In addition to the classical histological classification, breast carcinomas are classified into phenotypes according to hormone receptors (estrogen receptor-RE-/progesterone receptor-PR) and growth factor receptor (human epidermal growth factor receptor-HER2) expression. Luminal tumors (ER/PR-positive/HER2-negative) are present in older patients with a better outcome. However, patients with HER2-positive or triple-negative breast cancer (TNBC) (ER/PR/HER2-negative) subtypes still represent highly aggressive behavior, metastasis, poor prognosis, and drug resistance. Therefore, new alternative therapies have become an urgent clinical need. In recent years, anticancer agents based on natural products have been receiving huge interest. In particular, carotenoids are natural compounds present in fruits and vegetables, but algae, bacteria, and archaea also produce them. The antioxidant properties of carotenoids have been studied during the last years due to their potential in preventing and treating multiple diseases, including cancer. Although the effect of carotenoids on breast cancer during in vitro and in vivo studies is promising, clinical trials are still inconclusive. The haloarchaeal carotenoid bacterioruberin holds great promise to the future of biomedicine due to its particular structure, and antioxidant activity. However, much work remains to be performed to draw firm conclusions. This review summarizes the current knowledge on pre-clinical and clinical analysis on the use of carotenoids as chemopreventive and chemotherapeutic agents in breast cancer, highlighting the most recent results regarding the use of bacterioruberin from haloarchaea.

Personalized Diet in Obesity: A Quasi-Experimental Study on Fat Mass and Fat-Free Mass Changes.

Considering that the prevalence of overweight and obesity in Southeast of Spain is high, the aim of this work was to analyze the relation between the adherence to a personalized diet and the effectiveness of changes in the body composition in overweight and obese adults in this region. This quasi-experimental study presents the following selection criteria: attendance at the consultation between 2006 and 2012, subjects ≥ 19 years of age with overweight or obesity. In total, 591 overweight or obese individuals were involved in this study, attending 4091 clinic consultations in total. Most of the sample consisted of subjects who attended >3 consultations (>1.5 months), and were females aged 19-64 years who obtained clinically significant changes in fat mass (FM, ≥5%) and recommended changes in fat-free mass (FFM, ≥0%). Based on the results obtained and the experience gained from this research, the following recommendations are established: (i) record fat mass and fat-free mass index as a complement to body mass index; (ii) use FM and FFM to evaluate effectiveness of interventions with the aim of obtaining body composition changes; (iii) use personalized diet to achieve significant changes in FM and avoid non-recommended changes in FFM.