Endochitinase and Chitobiosidase Production by Marine Aeromonas caviae CHZ306: Establishment of Nitrogen Supplementation

Aeromonas caviae CHZ306, a marine-derived bacterium isolated from zooplankton, can use chitin (a polymer of a β-(1,4)-linked N-acetyl-D-glucosamine) as a carbon source. The chitin is hydrolyzed by chitinolytic enzymes, namely endochitinases and exochitinases (chitobiosidase and N-acetyl-glucosaminidase). Indeed, the chitinolytic pathway is initiated by the coexpression of the enzymes endochitinase (EnCh) and chitobiosidase (ChB); however, few studies, including biotechnological production of these enzymes, have been reported, although chitosaccharide are helpful in several industries, such as cosmetics. This study demonstrates the potential to maximize the simultaneous EnCh and ChB production by nitrogen supplementation on culture media. Twelve different nitrogen supplementation sources (inorganic and organic) previously analyzed in elemental composition (carbon and nitrogen) were tested and evaluated in the Erlenmeyer flask culture of A. caviae CHZ306 for EnCh and ChB expression. None of the nutrients inhibited bacterial growth, and the maximum activity in both EnCh and ChB was observed at 12 h, using corn-steep solids and peptone A. Corn-steep solids and peptone A were then combined at three ratios (1:1, 1:2, and 2:1) to maximize the production. The high activities for EnCh (30.1 U.L−1) and ChB (21.3 U.L−1) were obtained with 2:1 corn-steep solids and peptone A, corresponding to more than 5- and 3-fold enhancement, respectively, compared to the control condition.


Introduction
Chitin, the second most abundant natural polymer, is a nitrogen-containing polysaccharide composed of a β-(1,4)-linked N-acetyl-D-glucosamine (GlcNac) [1,2]. Chitin is widely distributed as a structural component of crustaceans, insects, and other arthropods and as a component of most fungi and some algae cell walls. Approximately 75% of the total weight of shellfish, such as shrimp, crabs, and krill, is considered waste, and chitin comprises 20-58% of the dry weight of the said waste [3]. Annually, more than 1000 metric tons of chitin are synthesized in the aquatic biosphere and deposited in marine sediment; thus, chitinolytic bacteria efficiently degrade this material to maintain the balance in the marine ecosystem [1,4]. Bacteria can synthesize chitinases to hydrolyze chitin as a carbon, nitrogen, and energy source, consequently making possible the recycling of chitin in the environment [2,5]. The chitinases (EC 3.2.11.14) are an efficient group of multiple enzymes classified into glycoside hydrolase families [6,7]. The chitin degradation chitin derivative production, the genome of Aeromonas caviae CHZ306 was sequenced, and its draft genome sequence was deposited at DDBJ/ENA/GenBank under the accession number MDSC01000000 [33]. The strain was grown on colloidal α-chitin agar plates at 28 • C for 96 h, according to the method described by Souza et al. (2009) [32]. To prepare the pre-inoculums, approximately five colonies were added to a 250 mL Erlenmeyer flask containing 100 mL of sterile colloidal chitin broth and incubated in an orbital incubator at 180 rpm and 28 • C for 24 h to obtain a cellular concentration of approximately 5.0 × 10 5 CFU.mL −1 .

Evaluation of Nitrogen Supplementation in Bacterial Growth and Chitinase Production
The ability of A. caviae CHZ306 to utilize inorganic and organic compounds as a supplementary nitrogen source was tested using the mineral-salt medium described below, in which either inorganic or organic compounds were present at a concentration of 0.2 g.L −1 . Colloid α-chitin (10 g.L −1 ) was used as the substrate. The broth medium base was composed of minerals and salts prepared to 1 L with: 0.2 g KH 2 PO 4 , 1.6 g K 2 HPO 4 , 0.2 g MgSO 4 .7H 2 O, 0.1 g NaCl, 0.01 g FeSO 4 .7H 2 O, and 0.02 g CaCl 2 .2H 2 O [32]. This mineral-based broth media was supplemented with 10 g.L −1 colloidal α-chitin and twelve different nitrogen sources listed in Table 1. The final pH medium was adjusted to 7.0. The cultures were carried out using a 250 mL Erlenmeyer flask containing 100 mL of medium with 1 mL pre-inoculum grown. The cultures were placed in an orbital incubator at 180 rpm, 28 • C, and samples (500 µL) were periodically collected for up to 96 h and evaluated for cell growth and chitinase activity.

Improvement of Endochitinase and Chitobiosidase Production by Nitrogen Source Mixture
The mineral-based medium described in the previous item included 10 g.L −1 of colloidal α-chitin, which was supplemented with mixtures of corn-steep solids and peptone A in 1:1, 1:2, and 2:1 proportions to reach a supplementation of 0.2 g.L −1 nitrogen (specific quantities are shown in Table 2). The cultures' conditions and analysis were carried out according to item 2.2.

Elemental Composition
Initially, the colloid chitin and complex nitrogenous sources were digested with concentrated HNO 3 and H 2 O 2 (30% w/w) with heating at 925 • C. Elemental composition for carbon (C) and nitrogen (N) was determined using an Optical Atomic Emission Spectrometer with Inductively Coupled Plasma (ICP-OES, Radial) model Arcos-Spectro ® (Ametek, Berwyn, PA, USA). Meanwhile, the C and N contents of chemically defined nutrients were calculated based on chemical composition and molecular mass.

Bacterial Growth
The drop plate method determined the cellular growth, expressed by colony-forming units per liter (CFU.L − ) [34]. Serially diluted samples (10 µL) were plated (in triplicate) on colloidal α-chitin agar plates at 34 • C for 96 h. * The required amount of each nitrogen source was calculated to achieve a 0.2 g.L −1 nitrogen supplementation in the mineral-based medium with the colloid α-chitin (10 g.L −1 ). C (carbon) and N (nitrogen). ** Final C:N ratio in the chitin-mineral medium after individual nutrient supplementation.

Chitinase Activity
Culture samples were centrifuged to collect the supernatant at 3000× g for 5 min at 4 • C. EnCh and ChB activities from the supernatant were determined by using the Chitinase Assay Kit (Sigma-Aldrich, Saint Louis, MO, USA), using 10 µL of supernatant and 90 µL of a specific substrate solution (1 mg.mL −1 of p-nitrophenol-triacylchitotriose [pNP-(GlcNAc) 3 ] or p-nitrophenol-diacetylchitobiose [pNP-(GlcNAc) 2 ]). The mixtures were incubated at 37 • C for 30 min; optical density was read at 405 nm using a microplate reader, model Spectramax ® plus 384 (Molecular Devices, Sunnyvale, CA, USA). The substrates [pNP-(GlcNAc) 3 ] and [pNP-(GlcNAc) 2 ] were used as substrates for EnCh and ChB, respectively. One unit (U) of EnCh and ChB was defined as the amount of each enzyme required to release 1.0 µmol of p-nitrophenol (pNP) from each substrate per minute.

Statistical Analysis
Experimental data for bacterial growth and enzymatic activity were presented as the average of three biological assays and expressed as the mean ± standard deviation (sd). Statistically significant differences (p < 0.05, 5% significance level) were determined by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post-test using the GraphPad Prism ® 7 software.

Evaluation of Nitrogen Supplementation in Bacterial Growth and Chitinase Production
A. caviae CHZ306 cultures were grown in an orbital shaker at 28 • C for 96 h to evaluate inorganic and organic nutrients that could potentially serve as growth substrates. A total of twelve nutrients were tested as nitrogen supplementation ( Table 1). As shown in Figure 1, none of the nutrients inhibited A. caviae CHZ306 growth, as the bacteria proliferated in all media tested, either with nitrogen supplementation from chemically defined (viz. M1-M4) or complex (viz. M5-M12) sources. Among all the media tested, corn-steep (M11) was far more favorable for bacterial growth and survival. This positive effect of corn-steep solids on bacterial growth was observed early, in the initial 12 h (3.37 × 10 9 CFU.L −1 ), remaining superior to all other media up to 96 h (3.4 × 10 10 CFU.L −1 ), as shown in Figure 2A,B. At 96 h, Figure 2B, corn-steep solids (M11) were the only source that achieved~10 10 CFU.L −1 growth, followed by M2 (ammonium acetate), M6 (meat extract), M7 (yeast extract), and M9 (peptone A), achieving~10 9 CFU.L −1 . Thus, comparing only the chemically defined media (M1 to M4), ammonium acetate supplementation (M2) enabled more remarkable microbial growth (1.42 × 10 9 CFU.L −1 ), presenting a similar profile of complex sources (viz. M7, M6, and M9). Besides, all nutrients tested, except ammonium nitrate (M3), provide superior bacterial growth than ammonium sulfate (M1, 1.32 × 10 8 CFU.L −1 ), commonly used in A. caviae cultivation [9,34,35]. The data obtained show that A. caviae CHZ306 utilized a variety of inorganic and organic compounds, particularly corn-steep solids (2.8 g.L −1 ), as a supplementary source of nitrogen, carbon, and energy for its growth.
Simultaneously, the ability of the A. caviae CHZ306 strain to produce both EnCh and ChB enzymes in a chitin-containing medium supplemented with nitrogen sources was tested throughout the 96 h cultivation. The effects of nitrogen supplementation on chitinase production are shown in Figure 3. Overall, the maximum activity in both enzymes was observed at 12 h ( Figures 3A and 4), then activity gradually decreased in all tested media ( Figure 3B-H). The medium supplemented with ammonium sulfate (M1), previously used in our studies, showed maximum activity of 5.6 and 5.9 U.L −1 at 12 h for EnCh ( Figure 4A) and ChB ( Figure 4B) enzymes, respectively. The highest activity for EnCh and ChB at 12 h was obtained using peptone A-M9 (23.1 and 21.2 U.L −1 , respectively) and corn-steep solids-M11 (32.1 and 23.0 U.L −1 , respectively), followed by M6 (meat extract), M8 (peptone Bacto), M10 (peptone G), and M12 (tryptone) that showed similar enzymatic activity. Compared to the ammonium sulfate (M1), the basal medium, peptone A (M9), and corn-steep solids (M11) showed 4.1-and 5.8-fold enhancements in EnCh production and 3.6-and 3.8-fold in ChB production, respectively. Notably, although M2 (ammonium acetate) and M7 (yeast extract) promoted bacterial growth, they were inefficient for EnCh and ChB production. In contrast, A. caviae CHZ306 grew less on M8, M10, and M12 media; however, it could express EnCh and ChB on these media. Table 2 presents three different mixtures of corn-steep solids (M11), and peptone A (M9) formulated to verify the interactions between these two selected nitrogen sources on A. caviae CHZ306 growth and chitinase production during 96 h, keeping constant the initial nitrogen supplementation of~0.2 g.L −1 .

Improvement of Selected Nitrogen Sources in Endochitinase and Chitobiosidase Production
As shown in Figure 5A, the A. caviae CHZ306 grew similarly in the three mixtures of corn-steep solids (M11), and peptone A (M9) studied, achieving~10 9 CFU.L −1 after 96 h of cultivation. Similar to the previous assay, the maximum activity for both enzymes was observed at 12 h, and their activities gradually reduced; notably, the EnCh activity decreased more than ChB over 96 h of cultivation. The EnCh production ( Figure 5B (ammonium acetate) and M7 (yeast extract) promoted bacterial growth, they were inefficient for EnCh and ChB production. In contrast, A. caviae CHZ306 grew less on M8, M10, and M12 media; however, it could express EnCh and ChB on these media.       Table 2 presents three different mixtures of corn-steep solids (M11), and peptone A (M9) formulated to verify the interactions between these two selected nitrogen sources on A. caviae CHZ306 growth and chitinase production during 96 h, keeping constant the initial nitrogen supplementation of ~0.2 g.L −1 .

Improvement of Selected Nitrogen Sources in Endochitinase and Chitobiosidase Production
As shown in Figure 5A, the A. caviae CHZ306 grew similarly in the three mixtures of corn-steep solids (M11), and peptone A (M9) studied, achieving ~10 9 CFU.L −1 after 96 h of cultivation. Similar to the previous assay, the maximum activity for both enzymes was observed at 12 h, and their activities gradually reduced; notably, the EnCh activity decreased more than ChB over 96 h of cultivation. The EnCh production ( Figure 5B

Discussion
In this study, we determined the nitrogenous compounds as potential substrat the growth of A. caviae CHZ 306, particularly in a chitin-containing mineral-based dium. A total of twelve different inorganic and organic nutrients, representing chem defined and complex sources, serve as substrates for bacterial growth. In general, the position of the culture medium (including nitrogen sources) significantly influenc cell growth, physiology, and biochemical pathways of microorganisms [36,37]. Thu enzyme production varies depending on the nitrogen source; in some cases, there m a preference for a particular type: nitrogen-rich sources are more rapidly assimilated nitrogen-poor sources due to the efficiency of transport systems [24,29,38]. In add the microorganisms can extract nitrogen from a broad spectrum of organic and inor substrates [27]. Therefore, screening and selecting the most suitable nitrogen sourc cultivation is essential to maximize product formation.
Ammonium sulfate (M1) and ammonium nitrate (M3) repressed the growth an tinase production, although ammonium ions could be directly assimilated and act a nors of amino acid synthesis [39]. In M1, the decrease in biomass production is comm caused by acidification by anion sulfate in media culture [40]. However, ammonium

Discussion
In this study, we determined the nitrogenous compounds as potential substrates for the growth of A. caviae CHZ 306, particularly in a chitin-containing mineral-based medium. A total of twelve different inorganic and organic nutrients, representing chemically defined and complex sources, serve as substrates for bacterial growth. In general, the composition of the culture medium (including nitrogen sources) significantly influences on cell growth, physiology, and biochemical pathways of microorganisms [36,37]. Thus, the enzyme production varies depending on the nitrogen source; in some cases, there may be a preference for a particular type: nitrogen-rich sources are more rapidly assimilated than nitrogen-poor sources due to the efficiency of transport systems [24,29,38]. In addition, the microorganisms can extract nitrogen from a broad spectrum of organic and inorganic substrates [27]. Therefore, screening and selecting the most suitable nitrogen source for cultivation is essential to maximize product formation.
Ammonium sulfate (M1) and ammonium nitrate (M3) repressed the growth and chitinase production, although ammonium ions could be directly assimilated and act as donors of amino acid synthesis [39]. In M1, the decrease in biomass production is commonly caused by acidification by anion sulfate in media culture [40]. However, ammonium sulfate was the most favorable nitrogen source to produce chitinase by Aeromonas sp. JK1 [31], and some authors also reported that Aeromonas species could degrade nitrates to nitrites [14]. Moreover, the efficient bacterial growth reached using ammonium acetate (M2) is possible since the salt dissolution in pure water produces a neutral pH solution due to the acidity of NH 4 + (pK a 9.25), which exactly balances the basicity of acetate (pK b 9.25) [41]. In general, inorganic nitrogen sources have an inefficient impact compared to organic sources [42]. However, urea (M4) presents low chitinase production even though the uptake is not protoncoupled and does not cause medium acidification in contrast to ammonium salts [19,40]. However, other Aeromonas species, such as A. hydrophila, can assimilate significant amounts of urea and nitrogen from ammonium through oxidation by glutamate synthase, while nitrate is utilized through nitrate reductase [43].
In the case of complex nutrients (viz. M5-M12), they could be assimilated by two independent pathways, such as a deamination reaction releasing ammonia or a transamination reaction, where the nitrogen is transferred to the acceptor substrate (α-ketoacid) [27]. Casaminoacid (M5) and yeast extract (M7) had a repressive effect on chitinase production. M5 is an acid hydrolysate of casein containing amino acids and short peptides, although it is adequate for media culture with minimal nitrogen requirements [42,43]. While M7 contains essential nutrients to satisfy nutritional requirements (i.e., carbohydrates, proteins, amino acids, vitamins, and trace elements) [44][45][46], some reports indicated no effect on chitinase production by A. hydrophila HS4 [3] and Aeromonas sp. JK1 [31]. M7 showed high cell growth and is considered, along with M11, the best source of organic nitrogen for biomass production. However, it is worth mentioning that the metabolic pathways of enzyme synthesis are much slower than the biomass production routes. [20,36]. On the other hand, meat extract (M6), peptone Bacto (M8), peptone G (M10), and tryptone (M12) showed significant chitinase production and growth at 12 h of culture. These results are possible due to composition; for example, M7 is a mixture of peptides and amino acids, nucleotide fractions, organic acids, minerals, and some vitamins [44,47,48]; M8 contains essential amino acids, low molecular peptides, trace elements, and vitamins [44,49]; M10, a pancreatic digest of gelatin, presents higher proline residues [44,50]; and M11, a pancreatic digest (i.e., trypsin hydrolysate) of casein, contains oligopeptides in different lengths and smaller amounts of amino acids and carbohydrates [44,45].
In our findings, peptone A (M9) and corn-steep solids (M11) were the most favorable nitrogen sources for simultaneous EnCh and ChB production, probably because these nutrients provide elements necessary for synthesizing chitinase enzymes. In this sense, both nitrogen sources were selected to continue evaluating chitinase production. M9 is a pancreatic digest of animal tissue and contains a mixture of small and large peptide sizes [44,49,50]. Thus, the nutritional support of peptones and derivates highly depends on the specific peptide (i.e., length and sequence) and free amino acid composition [20]. Moreover, M11 is a soluble solids by-product of the corn milling industry used for microbial growth, providing amino acids, vitamins, minerals, and growth factors [51,52]. The presence of corn-steep solids was also essential to increase the production of the L-asparaginase enzyme by Leucosporidium scottii L115 [37].
Comparative data from chitinase production by Aeromonas species are presented in Table 3. Some authors indicated that culture medium containing complex nutrients, e.g., peptones, tryptone, yeast extract, and malt extract, despite unknown composition, are cost-effective for processes due to their ability to support maximum product yield and performance, including for large-scale fermentations [36,49].
It is known that the composition of the culture medium affects the final cost of a bioprocess, directly affectingmicrobial growth and product formation [37]. The organic nitrogen from complex sources could be metabolized by cells directly, promoting chitinase production due to its high concentration of most amino acids and growth factors [53]. In this sense, manipulating medium components and concentrations is essential in microbial development because the range of nutrients required depends upon their metabolism [51,54]. Most microorganisms can sense qualitative and quantitative nutrient changes, improving their utilization in competitive environments [21].
Here, maximum chitinase (EnCh and ChB) yields were obtained in the mixture medium M11-M9 (2:1). Indeed, the enzyme production using the mixture of nitrogen sources could be higher than a single nitrogen source [36]. Furthermore, because some organic nitrogen sources are expensive in industrial bioprocess, medium costs can be reduced by replacing them entirely or partially with a less expensive component [50,51]. Medium M11-M9 (2:1) also presents a high C:N ratio, although Arous et al. (2016) [55] indicated that high C:N ratios are favorable for the accumulation of carbohydrates and intracellular lipids instead of enzyme production. This result might be the consequence of channeling more carbon for cell growth and chitinase production.
Moreover, it is suggested that enzyme production was obtained by using quickly metabolized nitrogen sources. Although no significant differences were observed in bacterial growth and chitinolytic activity between medium M11 and the mixture M11-M9 (2:1), the availability of commercial corn-steep solids is reduced and could hinder their acquisition at a large scale. In this sense, partial replacement with peptone A is a beneficial strategy.
Finally, A. caviae CHZ306 represents an exciting source of enzymes because it contains different genes encoding chitinases [33]. Moreover, in opposition to the chemical process for chitin degradation, enzymatic hydrolysis is a more sustainable alternative because it does not require the presence of toxic compounds or excessive wastewater generation [56]. The chitin derivatives such as chitosan and chitosaccharides present biological and physicochemical properties interesting for several applications, including biotechnology, biomaterials, food, medicine, cosmetics, wastewater treatments, etc. [56,57]. In this sense, using purified EnCh or ChB in a specific chitin-degradation step and controlling the length of the chitosaccharide would allow the definition of molecules and the evaluation of their biological activity. Additionally, a deep study of the biochemical properties of both enzymes is recommended to obtain a detailed understanding of reaction mechanisms. It is also possible to take advantage of the synthetic capacity of hydrolases in unconventional media (i.e., the synthesis of complex oligosaccharides and polysaccharides). * The values were estimated based on the graphics reported in the works; nr = not reported. Ench: endochitinase, and ChB: chitobiosidase.

Conclusions
Medium composition improvement is relevant in developing a commercial fermentation process affecting microbial growth and product yield. Based on our study, complex nitrogen sources play an essential role in chitinase production. The supplementation of peptone A (M9) and corn-steep solids (M11) as sole nitrogen sources increased EnCh and ChB production by at least 4.1-and 3.6-fold, respectively. In this sense, the organic nitrogen sources, corn-steep solids, and peptone A (at a ratio of 2:1), have been shown to stimulate the cell growth of A. caviae CHZ306 and to promote the high synthesis of EnCh (30.1 U.L −1 ) and ChB (21.3 U.L −1 ), in comparison with basal medium, corresponding to more than 5and 3-fold enhancement, respectively. Further assays should focus on optimizing culture conditions and process implementation in automated bioreactor systems.