Bioprospecting for Novel Halophilic and Halotolerant Sources of Hydrolytic Enzymes in Brackish, Saline and Hypersaline Lakes of Romania

Abstract

Halophilic and halotolerant microorganisms represent promising sources of salt-tolerant enzymes that could be used in various biotechnological processes where high salt concentrations would otherwise inhibit enzymatic transformations. Considering the current need for more efficient biocatalysts, the present study aimed to explore the microbial diversity of five under- or uninvestigated salty lakes in Romania for novel sources of hydrolytic enzymes. Bacteria, archaea and fungi were obtained by culture-based approaches and screened for the production of six hydrolases (protease, lipase, amylase, cellulase, xylanase and pectinase) using agar plate-based assays. Moreover, the phylogeny of bacterial and archaeal isolates was studied through molecular methods. From a total of 244 microbial isolates, 182 (74.6%) were represented by bacteria, 22 (9%) by archaea, and 40 (16.4%) by fungi. While most bacteria synthesized protease and lipase, the most frequent hydrolase produced by fungi was pectinase. The archaeal isolates had limited hydrolytic activity, being able to produce only amylase and cellulase. Among the taxonomically identified isolates, the best hydrolytic activities were observed in halotolerant bacteria belonging to the genus Bacillus and in extremely halophilic archaea of the genera Haloterrigena and Halostagnicola. Therefore, the present study highlights that the investigated lakes harbor various promising species of microorganisms able to produce industrially valuable enzymes.

Keywords: extracellular hydrolases; extreme environments; extremozymes; halophiles; halophilic archaea; halotolerant bacteria; hydrolytic enzymes; hypersaline lakes; salt-tolerant enzymes.

Figure 1

Geographic locations and overview photos of the five studied lakes. Three sites per lake were sampled: Lake Amara (AM) (44°36′20.9″ N, 27°19′39.6″ E; 44°36′23.8″ N, 27°19′35.9″ E; 44°36′23.0″ N, 27°19′14.9″ E), Lake Balta Alba (BA) (45°17′38.1″ N, 27°20′54.1″ E; 45°17′37.1″ N, 27°20′55.6″ E; 45°17′40.7″ N, 27°20′51.8″ E), Lake Caineni Bai (CB) (45°11′00.3″ N, 27°20′01.4″ E; 45°11′00.7″ N, 27°19′59.2″ E; 45°10′54.8″ N, 27°19′28.2″ E), Movila Miresii Salt Lake (MM) (45°13′16.0″ N, 27°38′25.8″ E; 45°13′15.4″ N, 27°38′31.6″ E; 45°13′16.0″ N, 27°38′20.1″ E), Braila Salt Lake (BSL) (45°12′57.6″ N, 27°54′38.4″ E; 45°12′58.8″ N, 27°54′37.7″ E; 45°12′58.4″ N, 27°54′40.3″ E).

Figure 2

The abundance, expressed as colony-forming units (CFU) per 1 mL of water (A) or 1 g of wet sediment (B), of the cultured fractions of halophilic/halotolerant microbial communities inhabiting the five investigated lakes. Bars show the differences between the three sampling sites. ND = Not Determined.

Figure 3

Relative abundances of halotolerant and halophilic microorganisms isolated from the five investigated lakes. The numbers in the bar graphs indicate the number of isolates.

Figure 4

Relative abundances of microbial hydrolase producers recovered from the five investigated lakes. The numbers in the bar graphs indicate the number of isolates that produced a particular enzyme.

Figure 5

Heat maps showing the ability of the Bacterial isolates—taxonomically identified on the basis of the 16S rRNA gene sequence analysis—to grow at different salt concentrations and produce extracellular hydrolytic enzymes.

Figure 6

Heat maps showing the ability of the Archaeal isolates—taxonomically identified on the basis of the 16S rRNA gene sequence analysis—to grow at different salt concentrations and produce extracellular hydrolytic enzymes.