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Novel industrial applications from salt loving extremophiles

Bob Grinshpon

Graduate Student Bob GrinshponI'm on ScienceSeeker-Microscope

This blog will review two recent publications that explore environmentally friendly advances in biotechnology by exploiting halophilic organisms from the family Halobacteriaceae. Halophiles are found in all kingdoms of life. They employ two different survival mechanisms to cope with their typically inhospitable environment. The first strategy, ‘organic solutes in,’ excludes external salt from the cytoplasm, and synthesizes osmolytes to balance the turgor pressure with the
environment. The second survival mechanism, ‘high salt in,’ is less common, and requires that the entire proteome adapt to high salt conditions. Halobacteriaceae consist of members that strictly
use the second strategy. The similarity between the two papers ends there, but each approach is
interesting in its own right.

In the first paper (1), they characterize β-galactosidase (bga gene) from the recently discovered polyextremophile, Halorubrum lacusprofundi, and assess it for potential use as an extremozyme. β-galactosidase is involved in the breakdown of β-galactosides into monosaccharides. It is noted for its use in production of lactose free milk. However, it is assumed that lactose is not typically available in H. lacusprofundi’s environment, and the bga gene is found clustered with other genes that suggest their role is in the breakdown of plant polymers.

The story behind H. lacusprofuni begins in 1956, when Russian scientists set up a laboratory at Lake Vostok Anarctica. It is believed to be Antarctica’s largest sub-glacial lake, and was left unexposed for at least 15 million years. Drilling began in 1990 and they reached the lake in February 2012, nearly 4,000 meters down. The salt concentrations of the lake can reach 4.0 molar, and freezing point depression is responsible for the -3◦ Celsius average temperature. An organism adapted to these conditions can provide insight into protein engineering by probing the structure and sequence for trends.

Lake Vostok Antarctica

Lake Vostok Antarctica

Consistent with green science trends, cold adapted enzymes decrease energy costs, and using extremozymes versus hazardous organic solvents reduces waste production. In order to be studied in more detail H. lacusprofuni bga was cloned and overexpressed in a genetically tractable host, Halobacterium sp. NRC-1. The proof of ability to overexpress twenty fold over the wild type exhibits potential for industrial scale production.

Their results show that β-galactosidase is still active as low as -5◦ Celsius, and more importantly, it is not immediately denatured in organic solvents. At high salinity, water is sequestered in hydrated ionic structures, limiting the water available for protein hydration.

Similarly, organic solutions have limited water availability. Mesophilic enzymes typically lose their activity in organic solvents, so the future of biofuel production may rely on a cocktail of engineered extremozymes to convert biomass into usable fuel. Concluding thoughts of this paper claim β-galactosidase to be a platform for improving our understanding of biocatalysts under limited water conditions.

The second paper (2) uses a completely different approach to utilizing halophilic organisms. Polyhydroxyalkanoates (PHAs) are biodegradable polymers that are potential substitutes for petrolchemical derived plastics. PHAs are synthesized by several microorganisms as reserves of carbon and energy when the absence of essential nutrients limits cellular growth. There are several different flavors of PHAs depending on the starting carbon source and the specific organism.

PHB is the more common product, but some organisms are capable of synthesizing a co-polymer, PHBV, which maintains enhanced mechanical properties. Haloferax mediterranei is a halophilic microorganism that is able to produce PHBVs up to 85% of its body mass in dry weight. Industrial production of PHAs is currently carried out by recombinant E. coli at a rate of 4.63 grams PHB per liter per hour. This is significantly higher than H. mediterranei’s 0.71 grams per liter per hour.

Biodegradable Biopolymers

Biodegradable Biopolymers

The goal of this paper was to improve the rate of production of PHBV by H. mediterranei,
because using H. mediterranei has many advantages to industrial production. Recovery of material can account for 40% of production costs, and since H. mediterranei uses the ‘high salt in’ survival strategy, lysing their cells is easily accomplished by putting them in water. Contamination and the costs of sterilization are reduced since high salt culture conditions inhibit the growth of other microbes. Lastly, H. mediterranei is a garbage disposal of cheap carbon sources. Although production rate maybe slower, the cost of production is 2.80 Euros per kilogram, versus 4.00 Euros per kilogram from E. coli.

One inherent disadvantage of H. mediterranei is the production of exopolysaccharide (EPS). EPS is also a byproduct of PHBV production in H. mediterranei. EPS is excreted into the medium and confers a smooth and wet characteristic to the colony. Accumulation of EPS increases the viscosity of the medium, and increases the foaming propensity. They located the gene cluster involved in EPS biosynthesis and knocked it down. The result of this knock down redirected the surplus carbon source to PHBV production. The mutant H. mediterranei increased total production of PHBV by nearly 20%, and nearly eliminated the use of anti-foaming agent. The decreased viscosity of the culture altered its redox state by increasing the level of dissolved oxygen. This also allowed for concentration of PHBV accumulation to increase from 0.7 g/L to 3.4 g/L. The future prospects for PHA production will involve marginally improving the output level until supply can reach the demand at a reasonable cost.

All in all, the discovery and exploitation of halophilic microorganisms has far reaching benefits to industrial sustainability. The extreme environments that they hail from propagate unique characteristics that can be exploited for technological advancement. Interestingly, the name Halobacteriaceae was coined in the 1970’s before the discovery of the domain archaea. This only shows you how shallow our understanding is of these organisms, and may infer that there is great potential for future applications in this field.

References:

1. Karan R., Capes M.D., DasSarma P. & DasSarma S. (2013). Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi, BMC Biotechnology, 13 (1) 3. DOI:

2. Zhao D., Cai L., Wu J., Li M., Liu H., Han J., Zhou J. & Xiang H. (2013). Improving polyhydroxyalkanoate production by knocking out the genes involved in exopolysaccharide biosynthesis in Haloferax mediterranei, Applied Microbiology and Biotechnology, 97 (7) 3027-3036. DOI: