Haloarchaea: History
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Subjects: Environmental Sciences
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Samy Selim

Haloarchaea has the capability of producing significant concentrations of polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), and polyhydroxyvalerate (PHV) when growing under a specific nutritional status. The growth of those microorganisms at the pilot or industrial scale offers several advantages compared to that of other microbes that are bioplastic producers.

  • bioplastics
  • polyhydroxyalkanoates (PHA)
  • polyhydroxybutyrate (PHB)
  • polyhydroxyvalerate (PHV)
  • haloarchaea

1. Introduction

Large-scale PHA/PHB production is an expensive process involving in most cases the use of organic solvents during the downstream processes (consequently, they are considered non-environmental biotechnological-based approaches). Mesophilic bacteria are vulnerable to contamination compared to extremophiles, and it is required to select robust production strains capable of growing in extreme conditions in which most of organisms could not proliferate. This is an important aspect to consider when designing large-scale biotechnological approaches. Consequently, extremophiles have been recently identified as the most promising microorganisms to produce PHA, reducing production costs [1]. Specifically, halophilic microbes show many advantages over non-halophilic microbes. Some of the main advantages are as follows: cell lysis is easy by exposing the cells to distilled water; many haloarchaeal species show fast growth compared to other microbial strains; sterilization is not essential because most common microbes used in lab (mesophiles) cannot grow under salty conditions; and, finally, waste materials like brines or salty waste waters from other processes could be used as culture media, thus minimizing the cost of the PHAs production.

Most halophilic archaea described to date can synthesize PHBV from structurally unrelated carbon sources, including starch, glucose, and glycerol [2]. Several studies show PHA production by haloarchaea genera, such as Halococcus, Haloferax, Halorubrum, Halobacterium, Natronobacterium, Natronococcus, Halopiger, or Haloarcula [3][4][5][6]. The genus Haloferax is of special interest due to its growth rate and high PHA productivity, the high quality of this PHA, and its substrate spectrum [7].

2. Advantages of Using Halophilic Microbes That Are Able to Produce PHA and PHB. Haloarchaea as Case of Study

2.1. Haloarchaeal Species Capable of Producing PHA, PHB and PHV

PHA accumulation in haloarchaeal cells was firstly described from Halobacterium sp. (currently known as Haloarcula marismortui) by Kirk and Ginzburg (1972) [8], half a century after the discovery of PHA by Lemoigne (1923) [9]. Since that time, more PHA-accumulating haloarchaea have been described. Fernandez-Castillo et al., in 1986 [10], found PHB in three species of the Haloferax genus and (Hfx. mediterranei, Hfx. volcanii, Hfx. gibbonsii) and in Haloarcula hispanica. Shortly after this work, accumulation of PHA was also observed in two more species of Haloarcula genus (Har. vallismortis and Har. japonica) [11][12]. Halopiger aswanensi was subsequently found to accumulate PHB for around 53% of its cell dry weight by using n-butyric acid and sodium acetate as sources of carbon [13][14]. Two species of Haloquadratum walsbyi as well as other species belonging to the following genera have been described as PHA producers: Halostagnicola, Haloterrigena, Halobiforma, Haloarcula, Halobacterium, Halocococcus, Halorubrum, Natrinema and haloalkaliphiles that include Natronobacterium and Natronococcus [3][15][16][17][18]. The third most abundant species in Antarctica’s Deep Lake, Halorubrum lacusprofundi, was recently confirmed to be a PHA-like granules-producer at low temperatures [19].

Hfx. Mediterranei is probably the most preferred PHA producer among all the haloarchaeal strains due to its high growth rate, metabolic versatility, genetic stability, and effective transformation system [20]. Many studies show that Hfx. Mediterranei can use many industrial and household wastes as carbon sources to synthesize PHA with significant productivity. In addition, PHA synthesized by Hfx. Mediterranei is a copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) from structurally unrelated substrates [21]. PHBV is a more versatile and economically favorable polymer than PHB [22]. Many species need precursor (3HV) for the synthesis of PHBV, while Hfx. Mediterranei can synthesize PHBV efficiently without such a precursor; therefore, this is another advantage of reducing the cost of its production [23]. Moreover, Hfx. Mediterranei is also capable of synthesizing poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) (PHBV4HB) in the presence of 4HB precursors in the medium (Y-butyrolactone) [24]. In brief, Hfx. Mediterranei has many advantages as a cell factory for the large-scale production of PHBV. Other strains, such as Har. hispanic, Hgm. borinquense, and Natrinema species, have also been found to accumulate PHBV by using unrelated substrates of carbon [25][26][27][28]; for instance, by using glucose as the sole source of carbon, Natrinema ajinwuensis accumulated PHBV with 13.93 mol% 3HV [29]. Moreover, Halogranum amylolyticum could efficiently accumulate PHBV at 20.1 mol% 3HV [30]. However, these haloarchaeal species, unlike Hfx. mediterranei, have disadvantages as a platform for further optimization of PHA production, such as low precursor for PHBV (3HV) content, slow growth rate, excess salinity requirements, or the absence of a manageable genetic transformation system. Nevertheless, these studies suggest that more haloarchaeal species with PHBV synthesis capability are being found [31].

2.2. PHA-Related Genes in Haloarchaea

Most of the haloarchaeal genomes are neither fully sequenced nor fully annotated [32]. This constitutes a significant limitation when bioinformatics approaches are used to explore genes and sequences related to specific metabolic processes. However, some molecules studies have been conducted regarding PHA synthesis.

The key enzyme involved in biosynthesis of PHA is PHA synthase catalyzing the polymerization of the hydroxyalkanoate monomer to produce PHA chains. PHA synthase in haloarchaea consists of two subunits, PhaC and PhaE, and belongs to class III. PHA synthase in halophilic bacteria, in contrast, consists of only the PhaC subunit and belongs to class I [31]. In haloarchaea, PHA synthase possesses some novel features, as shown below.

The first archaeal PHA synthase activity was detected by Hezayen and co-workers et al. (2002) from the halophilic archaeon called strain 56 [16]. PHA synthase was associated covalently with PHB granules, and the expression of the genes coding for it was only induced under nutritional conditions promoting PHB accumulation. However, molecular characterization of the enzyme was not possible due to lack of the sequence similarity. Using complete genome sequence of Har. marismortui, Han and co-workers identified and characterized PHA synthase genes [33]. Har. hispanica, a haloarchaeon phylogenetically close to Har. marismortui, was also analyzed in this study, finding highly homologous phaEC genes present in the genome and PHB production up to 9.9% (wt) [33]. Only phaC and phaE co-expression in the ΔphaEC mutant strain restored PHA accumulation of Har. hispanica. However, it has been found that the PhaC protein is stably attached to the PHA granules, whereas PhaE is not. This observation suggests that in Haloarcula species, the PhaC and PhaE subunits constitute a novel type of class III PHA synthase.

Likewise, PHA synthase of Hfx. mediterranei also consists of two subunits of PhaE and PhaC, and both proteins are constitutively expressed in nutrient-limited and nutrient-rich media [26]. phaE and phaC genes were also reported in Hgn. amylolyticum by Zhao et al. (2015) [30], and the PhaE and PhaC amino acid sequence showed 64% and 62% identity with Hfx. Mediterranei proteins, respectively. PhaC has been detected in Hrr. Lacusprofundi; this haloarchaeon produces PHA at low temperatures [19]. The molecular weight of PhaE and PhaC from haloarchaea was found to be 20 kDa and 50.1 to 58.5 kDa respectively, differing from that of the bacterial class III PHA synthase (40 kDa) [34]. Haloarchaeal PhaC consists of a longer C-terminal as compared to bacterial PhaC [3][34][17][19][21][23][26]. This longer C-terminus is essential for activation of PHA synthase because its truncation results in less PHA accumulation.

Furthermore, unlike the bacterial PhaE subunit, PhaE-box was present only in Haloarcula species and Halorhabdus utahensis DSM-12940, suggesting that this box is not so preserved in PhaEs. The phylogenetic analysis of PHA synthase from haloarchaea and bacteria clearly clustered them into two distinct domains [34]. This strongly suggests that PHA synthase from the haloarchaea is a novel subtype of class III PHA synthase. In addition to the phaC gene clustered with phaE, three additional phaC paralogues (designated as phaC1, phaC2, and phaC3) were described in Hfx. Mediterranei [34]. Although during PHBV accumulation the three additional genes were not transcribed, expression of PhaE from Hfx. mediterranei led to accumulation of PHBV with a varied 3HV content. Phylogenetic analysis based on PhaCs Hfx. mediterranei and PhaCs from other haloarchaeal species indicated that three additional PhaCs may have evolved through horizontal transfer from other sources and not directly from Hfx. Mediterranei PhaCs [31].

2.3. Effects of Cultivation Conditions on the Production of PHAs

Accumulation of PHAs as intracellular carbon and energy storage granules is one of the main strategies used by organisms that are PHA producers. This is in fact a stress response employed by many microorganisms and plants to adapt to their environment. Thus, an excess of carbon substrate and a deficit of other elements, such as nitrogen and phosphorus, are the nutrient-limiting stress conditions that induce the synthesis of PHA [35]. The carbon source accounts for up to 50% of production costs, and for that reason, great efforts have been made to identify nutritional conditions based on inexpensive raw materials, such as industrial waste streams, to find low-cost and feasible culture media to upscale in terms of mid–large-scale production of these biopolymers. Haloarchaea are very versatile microorganisms in terms of carbon sources for PHA production, and the waste streams utilized for that purpose include hydrolyzed whey from dairy industry, pretreated vinasse from the ethanol industry, olive mill wastewater, rice-based ethanol stillage, crude glycerol from the biodiesel industry, enzymatic extruded starch, date palm sugars, seaweed hydrolysate, and sugarcane bagasse, among others. Of note, haloarchaea from the genus Haloarcula sp. can accumulate up to 46.6% PHA in DCW using petrochemical wastewaters as a carbon source [36]. Most haloarchaea can synthesize the copolymer PHBV even without any external precursor, and they also contribute to reducing the production cost. The 3HV molar fraction in PHBV produced from glucose as the sole carbon source reached up to 9% in Haloferax mediterranei [22] and exceeded 20% in Halogeometricum borinquense E3 [25]. Moreover, the 3HV molar fraction can reach up to 99% in Haloferax mediterranei in the presence of direct precursors in the culture media, such as in the case of volatile fatty acids [37].

3. Conclusions

Microbiologically produced PHAs are viable candidates to replace conventional oil-based plastics. However, the production costs of this biopolymer are still considerably higher in comparison to those of traditional polymers. Important challenges should be overcome, including the costs of the carbon source as well as enhancing the production and extraction efficiency. In these terms, haloarchaea species provide the advantage of utilizing inexpensive carbon sources of industrial origin, lower energy requirements due to negligible sterility precautions, downstream processing without the use of any chemical solvent for cell lysis, the recyclability of process side-streams (spent fermentation broth and cell debris), and the production of 3HV-containing copolyesters from unrelated carbon sources. Promising findings regarding haloarchaea-related PHA production have been reported in recent years at the laboratory scale. However, very few works have provided an in-depth characterization at the pilot or semi-industrial scale, and marketable prototypes from haloarchaeal PHA and techno-economic assessments are scarce. What is needed now is to drive the upscaling of those promising processes at the lab-scale to boost the development of archaeal cell factories and illustrate their potential in the future of sustainable biotechnology.

This entry is adapted from the peer-reviewed paper 10.3390/md19030159

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