Sergey Y. But

This review provides a comprehensive overview of the current landscape in Bacillus spp. fermentation processes for high-value bioproducts such as proteases, amylases, and surfactin in lab-scale bioreactors, highlighting critical process features. An analysis of key findings over the past 5 years shows that an efficient fermentation requires customized bioreactor conditions dependent on the substrate, strain type, and the target product. Major challenges include overcoming foaming, ensuring oxygen mass transfer, and precisely balancing the growth and production phases. A combined approach using statistical modeling, such as response surface methodology, along with experimental validation in different bioreactor designs is recommended for the process optimization. This integrated strategy can significantly improve the efficiency, predictability, and overall success of Bacillus-based bioprocesses.

It has been shown that compatible solutes trehalose and N-acetylglutaminylglutamine amide (NAGGN) are accumulated in the cells of aerobic methylotrophs Methylobrevis pamukkalensis PK2^T, Methylobacterium nodulans ORS 2060^T, Hansshlegelia quercus Dub, and Methylopila capsulata IM1^T growing in a saline medium. Trehalose was found to be the dominant compound in the total compatible solute pools in these bacteria. Homologues of the enzymes responsible for the biosynthesis of NAGGN, N-acetylglutaminylglutamine synthetase (Ngg) and N-acetylglutaminylglutamyl amidotransferase (AsnO) were found in a number of genomes of methylotrophs belonging to the phylum Proteobacteria, and these genes were generally arranged into a single cluster also containing the gene for a putative aminopeptidase of the M42 family. Genomic analysis has shown that NAGGN-synthesizing methylotrophs encode up to three trehalose biosynthesis pathways. Cultivation of M. pamukkalensis PK2^T on methanol or mannitol in the presence of choline was accompanied by the reduced level of NAGGN and the lack of trehalose in the cells. The involvement of NAGGN and trehalose in the adaptation of methylotrophs to existence either under the conditions of high salinity or in association with plants is discussed. The discovery of a new spectrum of compatible solutes in aerobic methylotrophs expands our understanding of their metabolic diversity and opens up new potential applications.

Methylococcus capsulatus MIR is a strain of methanotrophic bacteria that is potentially suitable for producing feed protein and other value-added products from methane. Genomic analysis did not reveal in Mc. capsulatus MIR known pathways of C3-carboxylation, necessary for the replenishment of intermediates of the tricarboxylic acid cycle and the full functioning of metabolism. The pepc gene encoding PEP carboxylase in Methylomonas rapida 12 was introduced into Mc. capsulatus MIR cells on a plasmid under the control of a medium-strength promoter. Expression of heterologous PEP carboxylase led to an increase in the content of glutamate, glycine and lysine in the cells of the recombinant strain, but did not increase to the growth rate of the culture. Consequently, the introduction of heterologous PEP carboxylase, which carries out the C3-carboxylation reaction, helps to increase the nutritional value of the methanotroph biomass.

Methylococcus capsulatus MIR is a strain of methanotrophic bacteria that is potentially suitable for production of feed protein and other value-added compounds from methane. Genomic analysis did not reveal in Mc. capsulatus MIR any known pathways of C3-carboxylation necessary for the replenishment of the tricarboxylic acid cycle intermediates and the full-scale metabolic functioning. The pepc gene encoding PEP carboxylase in Methylomonas rapida 12 was introduced into Mc. capsulatus MIR cells on a plasmid under the control of a medium-strength promoter. Expression of heterologous PEP carboxylase led to an increase in the content of glutamate, glycine, and lysine in the cells of the recombinant strain, but did not increase the growth rate of the culture. Consequently, the introduction of heterologous PEP carboxylase, which carries out the C3-carboxylation reaction, helps to increase the nutritional value of the methanotroph biomass.

The biotechnology of converting methane to single-cell protein (SCP) implies using fast-growing thermotolerant aerobic methanotrophic bacteria. Among the latter, members of the genus Methylococcus received significant research attention and are used in operating commercial plants. Methylococcus capsulatus MIR is a recently discovered member of this genus with the potential to be used for the purpose of SCP production. Like other Methylococcus species, this bacterium stores carbon and energy in the form of glycogen, particularly when grown under nitrogen-limiting conditions. The genome of strain MIR encodes two glycogen synthases, GlgA1 and GlgA2, which are only moderately related to each other. To obtain glycogen-free cell biomass of this methanotroph, glycogen synthase mutants, ΔglgA1, ΔglgA2, and ΔglgA1ΔglgA2, were constructed. The mutant lacking both glycogen synthases exhibited a glycogen-deficient phenotype, whereas the intracellular glycogen content was not reduced in strains defective in either GlgA1 or GlgA2, thus suggesting functional redundancy of these enzymes. Inactivation of the glk gene encoding glucokinase also resulted in a sharp decrease in glycogen content and accumulation of free glucose in cells. Wild-type strain MIR and the mutant strain ΔglgA1ΔglgA2 were also grown in a bioreactor operated in batch and continuous modes. Cell biomass of ΔglgA1ΔglgA2 mutant obtained during batch cultivation displayed high protein content (71% of dry cell weight (DCW) compared to 54% DCW in wild-type strain) as well as a strong reduction in glycogen content (10.8 mg/g DCW compared to 187.5 mg/g DCW in wild-type strain). The difference in protein and glycogen contents in biomass of these strains produced during continuous cultivation was less pronounced, yet biomass characteristics relevant to SCP production were slightly better for ΔglgA1ΔglgA2 mutant. Genome analysis revealed the presence of glgA1-like genes in all methanotrophs of the Gammaproteobacteria and Verrucomicrobia, while only a very few methanotrophic representatives of the Alphaproteobacteria possessed these determinants of glycogen biosynthesis. The glgA2-like genes were present only in genomes of gammaproteobacterial methanotrophs with predominantly halo- and thermotolerant phenotypes. The role of glycogen in terms of energy reserve is discussed.

Considering the increasing interest in understanding the biotic component of methane removal from our atmosphere, it becomes essential to study the physiological characteristics and genomic potential of methanotroph isolates, especially their traits allowing them to adapt to elevated growth temperatures. The genetic signatures of Methylocaldum species have been detected in many terrestrial and aquatic ecosystems. A small set of representatives of this genus has been isolated and maintained in culture. The genus is commonly described as moderately thermophilic, with the growth optimum reaching 50 °C for some strains. Here, we present a comparative analysis of genomes of three Methylocaldum strains—two terrestrial M. szegediense strains (O-12 and Norfolk) and one marine strain, Methylocaldum marinum (S8). The examination of the core genome inventory of this genus uncovers significant redundancy in primary metabolic pathways, including the machinery for methane oxidation (numerous copies of pmo genes) and methanol oxidation (duplications of mxaF, xoxF1-5 genes), three pathways for one-carbon (C1) assimilation, and two methods of carbon storage (glycogen and polyhydroxyalkanoates). We also investigate the genetics of melanin production pathways as a key feature of the genus.

Considering the increasing interest in understanding the biotic component of methane removal from our atmosphere, it becomes essential to study the physiological characteristics and genomic potential of methanotroph isolates, especially their traits allowing them to adapt to elevated growth temperatures. The genetic signatures of Methylocaldum species have been detected in many terrestrial and aquatic ecosystems. A small set of representatives of this genus has been isolated and maintained in culture. The genus is commonly described as moderately thermophilic, with the growth optimum reaching 50oC for some strains. Here, we present a comparative analysis of genomes of three Methylocaldum strains—two terrestrial M. szegediense strains (O-12 and Norfolk) and one marine strain Methylocaldum marinum (S8). The examination of the core genome inventory of this genus uncovers significant redundancy in primary metabolic pathways, including the machinery for methane oxidation (numerous copies of pmo genes) and methanol oxidation (duplications of mxaF, xoxF1-5 genes), three pathways for one-carbon (C1) assimilation, and two methods of carbon storage (glycogen and polyhydroxyalkanoates). We also investigate the genetics of melanin production pathways, as a key feature of the genus.

The genome of aerobic methanotroph Methylococcus capsulatus Bath possesses genes of three biochemical pathways of C1-carbon assimilation: the ribulose monophosphate cycle, the Calvin–Benson–Bassham cycle, and the partial serine cycle. Numerous studies have demonstrated that during methanotrophic growth cells of Methylococcus capsulatus Bath express key enzymes of these routes. In this study, the role of the serine cycle key enzymes, serine-glyoxylate aminotransferase (Sga) and malyl-CoA lyase (Mcl) in metabolism of Methylococcus capsulatus Bath was investigated by gene inactivation. The Δsga mutant obtained by double homologous recombination showed a prolonged lag phase, and after the lag period, the growth rate became similar to that of the wild type strain. The elevated intracellular levels of glutamate, serine, glycine, alanine, methionine, leucine, and succinate suggested significant metabolic changes in the mutant cells. Deletion of the mcl gene resulted in very poor growth and glycine only partially improved growth of the mutant strain. Cells of Δmcl mutant possess lower content of histidine, but enhanced level of alanine, leucine, and lysine than those of the wild type strain. Our data imply the importance of the serine cycle enzymes in metabolism of the methanotroph as well as relationships of the three C₁ assimilation pathways in the gammaproteobacterial methanotrophs.

The biochemical routes for assimilation of one-carbon compounds in bacteria require many clarifications. In this study, the role of malyl-CoA lyase in the metabolism of the aerobic type I methanotroph Methylotuvimicrobium alcaliphilum 20Z has been investigated by gene inactivation and biochemical studies. The functionality of the enzyme has been confirmed by heterologous expression in Escherichia coli. The mutant strain lacking Mcl activity demonstrated the phenotype of glycine auxotrophy. The genes encoding malyl-CoA lyase are present in the genomes of all methanotrophs, except for representatives of the phylum Verrucomicrobium. We suppose that malyl-CoA lyase is the enzyme that provides glyoxylate and glycine synthesis in the type I methanotrophs supporting carbon assimilation via the serine cycle in addition to the major ribulose monophosphate cycle.

The indicator enzyme of the serine pathway of assimilation of reduced C₁ compounds, serine-glyoxylate aminotransferase (Sga), has been purified from three methane-oxidizing bacteria, Methylomicrobium alcaliphilum 20Z, Methylosinus trichosporium OB3b and Methylococcus capsulatus Bath. The native enzymes were shown to be dimeric (80 kDa, strain 20Z), tetrameric (~ 170 kDa, strain OB3b) or trimeric (~ 120 kDa, strain Bath). Sga from the three methanotrophs catalyse the pyridoxal phosphate-dependent transfer of an amino group from serine to glyoxylate and pyruvate; the enzymes from strains 20Z and Bath also transfer an amino group from serine to α-ketoglutarate and from alanine to glyoxylate. No other significant differences between the Sga from the three methanotrophs were found. The three methanotrophic Sga have their highest catalytic efficiencies in the reaction between glyoxylate and serine, which is in agreement with their function to provide circulation of the serine assimilation pathway.The disruption of the sga gene in Mm. alcaliphilum resulted in retardation of growth rate of the mutant cells and in a prolonged lag-phase after passaging from methane to methanol. In addition, the growth of the mutant strain is accompanied by formaldehyde accumulation in the culture liquid. Hence, Sga is important in the serine cycle of type I methanotrophs and this pathway could be related to the removal of excess formaldehyde and/or energy regulation.