Bioluminescence and Chitinase Production during Chitin Fermentation by Vibrio harveyi

Bioluminescence and Chitinase Production during Chitin Fermentation by Vibrio harveyi

Badireddy Madhusudana Rao , Floyd L. Inman III 1, 2 1 and Leonard D. Holmes 1 1. Sartorius Stedim Biotechnology Laboratory, Biotechnology Research and Training Center, University of North Carolina, North Carolina 28472, USA 2. Central Institute of Fisheries Technology, Visakhapatnam Research Centre, Andhra University, Visakhapatnam 530003, India

Received: December 18, 2012 / Accepted: February 05, 2013 / Published: May 30, 2013.

Abstract: Vibrio harveyi, like other luminescent bacteria, is capable of producing extracellular chitinases. Microbial chitinases are utilized to depolymerize chitin into chitooligosaccharides and N-acetylglucosamine for the acquisition of carbon and possibly nitrogen, needed for survival. For many luminous marine bacteria (Vibrio spp.), quorum-sensing is highly speculated to be responsible for bioluminescence; however, in terrestrial species (Photorhabdus spp.) luminosity seems to be controlled through unknown mechanism of phase variation. In the present work, the correlation between bacterial luminosity and chitinase production of

V. harveyi was studied. The utilization of bioluminescence could prove to be an easier and more convenient method to monitor chitin fermentations that employ luminous bacteria. Results from the fermentation study indicate that luminosity of V. harveyi inversely correlates with chitinase production. In other words, during chitin fermentation, chitinase production was seen to increase while luminosity decreased with respect to growth and growth conditions. Furthermore, the results also suggest that V. harveyi may utilize an alternate mechanism that can counter quorum-sensing mechanisms to ensure bacterial survival under deteriorating growth conditions. The inverse relationship observed in this study may lead to a basic understanding of monitoring and studying chitin fermentations and anti-quorum-sensing/phase variation mechanisms exhibited by luminous bacteria.

Key words: Vibrio harveyi, bioluminescence, chitinase, chitin fermentation.

1. Introduction bioluminescence in V. harveyi serves as DNA photoreactivation repair mechanisms and

Vibrio harveyi is a marine bacterium endowed with detoxification of toxic oxygen derivatives [4, 5]. the dual capability of producing both bioluminescence Bioluminescence in V. harveyi is controlled by the and enzymes for chitin degradation. These dual luxCDABE operon that is regulated by chemical attributes are well established in other bioluminescent autoinducers known as “quorum sensing” [6, 7]. bacteria such as Vibrio fischeri, Photobacterium Chitinases are extracellular enzymes that have leiognathi , Photorhabdus luminescens and Vibrio been detected to be produced by a wide variety of cholerae [1, 2]. Furthermore, the ecological organisms where the roles of chitinases in these significance between bioluminescence and chitinase organisms are diverse [8]. Chitin degradation is production remains intriguing and unknown. performed by a battery of chitinases that include Bioluminescence is energy intensive, where energy depolymerases, chitodextrinases, deacetylases, committed to luminosity accounts for 10% of the total N -acetylglucosaminidases, and chitosanases. metabolism [3]. Researchers have shown that Chitinases have a wide range of applications,

especially in the production of chitooligosaccharides Corresponding author: Leonard D. Holmes, Ph.D.,

associate professor, research fields: fermentation technology, and N-acetylglucosamine where they can be used as applied microbiology and biochemistry. E-mail: antibacterial agents and immunoenhancers [9].

len.holmes@uncp.edu.

Bioluminescence and Chitinase Production during Chitin Fermentation by Vibrio harveyi

Microbial chitinases can also be utilized for safe the release of N-acetylglucosamine (NAG) from chitin waste management of chitin-containing compounds.

[14]. Briefly, 0.5 mL of culture supernatant was added India alone generates 60,000-80,000 tons of to 2 mL of colloidal chitin (1.25%), incubated at chitinous wastes from shrimp processing [10].

25 °C for 2 h; placed in boiling water for 5 min and Researchers have found that the chitinolytic activity

cooled in chilled water. One unit of of bacteria on shrimp waste was much higher than

β-N-acetylglucosaminidase was added, incubated at chitinase activity of fungi [11]. V. harveyi is an ideal

25 °C for 30 min and centrifuged. 1 mL of supernatant chitinase-producing candidate as it also has the

was added to 2 mL of distilled water and 1.5 mL of ability to attach to chitinous materials [12]. The

3,5-dinitrosalicylic acid color reagent (DNS), placed present study evaluates the relationship between

in boiling water for 5 min, cooled to ambient bioluminescence and chitinase production to further

temperature and A 540 was measured. The amount of understand and monitor chitinase production in V.

NAG liberated was determined using the standard harveyi .

curve with known amounts of NAG. Chitinase from Streptomyces griseus was used as the positive control.

2. Materials and Methods

One unit (U) of chitinase is defined as the amount of

2.1 Organisms and Media chitinase that liberates 1 mg of NAG from chitin per hour at pH 6.0 at 25 °C in a two hour two-step

In this investigation, V. harveyi ATCC 14126 was reaction with β-N-acetylglucosaminidase. The utilized. Luria Bertani broth and/or agar containing quantity of chitinase produced was expressed as units 3% NaCl (LB) and LB containing 2% colloidal chitin

per liter (U/L).

(LBC) were utilized to study V. harveyi in vitro.

2.2.3 Quantification of Protein and Culture Density Colloidal chitin was prepared from shrimp shells Protein concentrations was determined as per according to Hsu and Lockwood [13]. All media (pH Bradford [15] employing the standard 3.1 mL protocol. 7.0), once inoculated, was incubated at 26 °C. Broth

A 595 was measured and protein concentration was cultures were also shaken at 150 rpm. obtained from a curve produced with bovine serum

2.2 Assay Protocols albumin. Culture density was obtained utilizing a visible spectrophotometer set to a 600 nanometer

2.2.1 Screening for Chitolytic Activity and

wavelength (A 600 ).

Bioluminescence

2.2.4 Culturing Conditions.

Bioluminescence, reported as relative luminosity LBC broth cultures (100 mL) of V. harveyi was (RLU), was measured from 1 mL culture aliquots incubated at 26 °C (room temperature) and stirred at utilizing a luminometer. Observations of luminosity 150 rpm on a platform orbital shaker. Samples were would be observed on LB and LBC agar in total obtained at regular intervals and used to measure darkness for approximately 15 minutes. Prescreening bioluminescence, optical density and chitinase

V. harveyi for chitinolytic activity on LBC

production.

agar plates incubated for three days at 26 °C. Verification of chitinase production will be

3. Results and Discussion

determined by clearings (halos) surrounding bacterial

3.1 Screening for Chitolytic Activity and colonies.

Bioluminescence

2.2.2 Quantification of Chitolytic Activity To quantify chitinase activity within the culture

V. harveyi grown on LBC agar did exhibit supernatant, the DNS assay was employed to measure

bioluminescence within 24 h at 26 °C; however,

Bioluminescence and Chitinase Production during Chitin Fermentation by Vibrio harveyi

chitinase production was not evident until the end of day three. A clear halo surrounding the colony is definitive of chitin degradation (Fig. 1a). Furthermore, bioluminescence was persistent along with chitinase activity (Fig. 1b), but was only observed at the periphery of actively growing cells.

3.2 Quantification of Chitolytic Activity Bioluminescence of V. harveyi exhibited a rapid

increase from 1.63  10 4 RLU to 1.59  10 6 RLU

from 4.3 to 6.3 hours post-inoculation (Fig. 2a). This increase in bioluminescence was observed during the deceleration growth phase of V. harveyi, as expected due to quorum sensing. However, due to the possibility of nutrient limitation, luminosity fell sharply within the next day and gradually decreased until the ending of the experiment.

Chitinase production by V. harveyi was first detected after one day of incubation and increased significantly by day four (Fig. 2b). No chitinase production was detected during day one which suggests that chitinase are not produced during exponential growth. However, our data depicts that chitinase production is induced in either late deceleration or early stationary phases. This data also suggests that chitinases may be produced

during conditions of nutrient limitation. When Fig. 2 Correlations between growth, luminosity and

chitinase production. (a) Luminosity ( ○) begins to increase

comparing luminosity to chitinase production as a

around 4 h (1.0  10 4 RLU) and achieves its maximum (1.0

function of time; an inverse relationship between

 10 7 RLU) at the end of the first day upon entrance into

the two can be seen starting after the first day (Fig.

the stationary phase of growth ( Δ); (b) Chitinase

production ( □) did not start to increase until the ending of day one (2 U/L) and continued to increase until stable (184 U/L) after 4 days during stationary phase of growth ( Δ); (c) The inverse relationship between luminosity ( ○) and chitinase production ( □) can be seen beginning day one and continues to the end of the experiment.

2c).