3.1. Isolation, screening, and identification of cellulolytic marine bacteria
The South China Sea (SCS) is vast, covering more than 2 million square kilometers, which is one of the largest marginal seas  with rich microbial resources in the world. There are a lot of seagrass ecosystems in SCS. Seagrass ecosystems are considered major blue carbon sinks, which occupy less than 0.2% of the area of the world’s oceans but are estimated to bury roughly 10% of the yearly estimated organic carbon burial in the oceans [40, 41]. Seagrass bed is also a marine ecosystem with high microbial diversity, and the cellulose is one of main organic matter . Therefore, we reason that bacteria with highly cellulolytic activates are likely to exist in these marine ecosystems. However, the knowledge of cellulolytic marine bacteria from Hainan seagrass beds is limited. Herein, our aim is to isolate cellulolytic marine bacteria for L. vannamei aquaculture from these areas to reduce its cost by hydrolyzing SB. 91 cellulolytic marine bacteria of 97 cellulolytic isolates were from seagrass bed ecosystems, indicating that seagrass beds can be used as an important place for screening the cellulolytic bacteria. These results indicated that Bacillus and Vibrio were the dominant cellulolytic bacteria in seagrass ecosystems. In addition, although we only obtained 6 cellulolytic bacteria from aquaculture environments, there were 4 strains belonging to Bacillus, indicting that Bacillus was also the dominant cellulolytic bacteria in aquaculture ecosystems.
Most cellulolytic enzymes isolated from mangrove were from bacteria belonging to the genera Micrococcus, Bacillus, Pseudomonas, Xanthomonas, and Brucella [43, 44]. The cellulolytic bacteria isolated from different areas have different properties. The cellulolytic bacterium from farm at Zhanjiang (Guangdong Province, China), Lactobacillus pentosus AS13, can effectively enhance the growth performance, feed utilization, digestive enzymes and disease resistance of L. vannamei . Geobacillus thermodenitrificans IP_WH1 from North West Himalayas could produced a thermotolerant cellulase . Therefore, these cellulolytic marine bacteria from the tropical marine environment may be also characterized by potential use.
So far, more researches on mesophilic and thermophilic cellulases have been reported, while fewer works were on low-temperature cellulase. Low-temperature enzymes can not only ensure high-efficiency enzyme reactions at low-temperature, but also inactivate enzymes at low-temperature, thereby saving energy and cost in the production process. The probability of developing low-temperature cellulase from terrestrial organisms is much lower than that from marine organisms. The marine environment is very unique. Marine microbial enzymes are usually identified by cold tolerance and salt-alkali resistance, and have unique application prospects compared with terrestrial microbial enzymes. Therefore, the cellulolytic marine bacteria from marine environments may be have more potential applications than those from terrestrial, particularly on mariculture.
3.2. Cellulolytic activity of cellulolytic marine bacterial strains
Cellulases are mainly divided into endoglucanases, exoglucanases and β-glucosidases, and the three enzymes act synergistically in cellulose hydrolysis . The cost of cellulolytic enzymes is a major factor in the hydrolysis of lignocellulosic materials to fermentable sugars . Currently, various cellulolytic microorganisms were from fungi, such as Aspergillus [48, 49], Penicillium [50, 51], Trichoderma [30, 52-54], and Talaromyces [55-57], which required long production cycles. Compared with fungi, bacteria not only have a higher growth rate, but also produce cellulase in a short time. At the present study, the highest FPase activity of Bacillus MW-C57, 2.325 U/mL, was 18-fold higher than that of NAB37 from Haryana . Unfortunately, MW-C57 didn’t display HC. The FPase activity of other marine bacteria from this study areas was similar to that from Haryana . The FPase activities of our marine bacteria, 0.018 to 0.597 U/mL, were lower than that of fungi, for example, the FPase of T. harzianum belonging to fungi was in the range of 0.445–2.7 U/mL .
In this study, a total 28 cellulolytic marine bacterial strains with HC value greater than 2 were classified as high enzyme activity strains, which were used for further screening the safe cellulolytic marine bacteria for L. vannamei (Table 3). The cellulolytic marine bacterial with the largest HC value were isolated, namely MW-M5 (HC value=3.951), MW-M10 (HC value=3.752), and MW-M14 (HC value=3.505). The HC values of cellulose degrading bacteria from mangrove soils were ranged from 1.25 to 2.5 , which were very similar to that from flower stalks-vegetable waste co-composting system (HC values were ranged from 0.4 to 2.1) . HC values of cellulolytic bacteria from oil palm were ranged from 1.56 to 4.14 .
It is worth noting that there were 30 strains with the relative enzyme content (REC) more than 3 U/g in the supernatant of fermentation broth after 24 h according to the linear relationship between HC value and content of commercial cellulase (2.4 U/g), which were significantly higher than that of commercial cellulose. The method for quantitatively analyzing the detected enzymic production of the strains according to the standard curve of known enzyme is more intuitive, faster, and more effective, which will provide a new idea for the method of measuring enzymic activity of cellulolytic strains.
3.3. Screening of the safe cellulolytic marine bacteria for L. vannamei aquaculture
For the aquaculture of L. vannamei, the use of probiotics as a group of live bacteria that added directly to water has beneficial effects on organisms in culture, such as water bioremediation, improvement of digestion, intensification of the immune response, and inhibition of the growth of pathogenic bacteria . FAO has suggested the use of probiotics as a major means for the improvement of aquatic environmental quality . People usually use probiotics to replace antibiotics in the process of aquaculture of aquatic animals, which can not only restrain the multiplying of pathogen and enhance the immunity of cultured animals, but also improve the aquaculture environment. However, there is no report about the cellulolytic probiotics from the tropical marine environment in Hainan. Therefore, the indigenous cellulolytic probiotics from the Hainan tropical aquaculture system and the marine environment of SCS that are beneficial for L. vannamei aquaculture will not only ensure the safety and improve the production of white shrimp, but also reduce the cost of aquaculture by using the cheap SB as the only carbon source.
3.3.1. Safety of cellulolytic marine bacteria to L. vannamei
In order to be considered as a probiotic, the strain has to be non-toxic to the host. 26-test cellulolytic marine bacteria were safe for L. vanname (Table 3), which were agreed with the earlier reports where no mortality has been detected by Macrobrachium rosenbergii and L. vannamei with L. pentosus [45, 63]. In aquaculture water, the changes of salinity will affect the physiological function of microorganisms, which will eventually hinder their growth, survival, and food consumption . In the present study, we also detected the salinity tolerance and pH tolerance of these 26 cellulolytic marine bacterial strains (data not shown). There were 11 strains suitable for fresh water (0‰–10‰) (CFW-C6, CFW-C18, SBC-C23, MW-C44, MW-C79, MW-M10, MW-M13, MW-M14, MW-M15, MW-M19, and MW-M20) and 11 strains suitable for aquaculture water (15‰–30‰) (CFW-C7, CFW-C32, MW-C42, MW-C45, MW-C52, MW-C61, MW-C77, MW-C81, MW-M5, MW-M9, and MW-M17). MW-C58 was suitable for high salinity environment (15‰–30‰), while MW-C48, MW-C63, and MW-M4 were identified as halophilic strains. In addition, all of these bacteria can’t grow under over-acidification condition (pH<4), while some strains can grow under alkaline condition (pH 10). There were 13 strains with a wide range of acid-alkali resistance. These results will provide important information for the adaptation of these cellulolytic marine bacteria to industrial aquaculture applications.
3.3.2. Antibiotic susceptibility assay
In addition to the detection of animal safety, antibiotic susceptibility analysis is also an effective method for identifying potential probiotics. The abuse of antibiotics will destroy the balance of the original microbial flora in the breeding environment, resulting in drug resistance. At the present study, the main purpose was to screen the highly safe strains with low drug resistance. Most test cellulolytic marine bacteria were susceptible to chloramphenicol. Chloramphenicol is a broad-spectrum antibiotic, which has a strong inhibitory effect on Gram-positive and Gram-negative bacteria (Goldfarb, Doi, Rodriguez, 1981), and has been used in aquaculture industry since 1980s (Cravedi, Choubert, Delous, 1987; Reide, Siegmund, 1989; Rijkers, Teunissen, Van Oosterom, Van Muiswinkel, 1980). According to our results, the animals in the study environments had been threatened by penicillin, piperacillin, carbenicillin, cefalexin, streptomycin, spectinomycin, tetracycline, erythromycin, and azithromycin, while some antibiotics with relatively lower MAR index could be reasonably used in these environments, such as cefoxitin, ceftriaxone, chloramphenicol, and furazolidone. More importantly, Vibrio sp. WM-M19 displayed a lowest MAR index (0.1), which was resistant to only three antibiotics, indicating that this strain was safe and non-drug resistance for further use. Of course, antibiotic resistance pattern may vary depending on the geographical locations and selective pressure and these patterns change rapidly from time to time .
3.4. Determination of the SB decomposition of the safe cellulolytic bacteria
Although high-density aquaculture can bring high profits, the accumulation of high-protein bait and excrement will aggravate the deterioration of the aquaculture water . Residual bait and feces are the main sources of nitrogen and phosphorus during the pressing of aquaculture, while the relative lack of carbon will lead to water deterioration [3, 11-14]. However, the high cost of adding carbon source artificially limits its application of large-scale popularization. Therefore, it has become a research hotspot to find cheap carbon sources. Sugarcane, a tropical crop, requires special environment, such as, an optimum high temperature, availability of sunshine and higher rainfall for its growth. The extraction of juice from sugarcane generates a fibrous residue, SB. Normally one ton of sugarcane produces 280 kg of SB . SB is rich in fermentable components and usually composed of 25% lignin, 25% hemicelluloses and 50% cellulose [66, 67]. Sugarcane is a widely grown sugar crop in Hainan, China, and its SB is the main wastes of sugar industry. However, there were few reports on SB decomposition of cellulolytic marine bacterial strains in shrimp aquaculture. The cellulase producing strains from seagrass beds of mangrove displayed relatively higher SBase, such as MW-M19 (4.14 U/mL) and MW-C77 (3.746 U/mL), we reasoned that the cellulolytic bacteria isolated from the seagrass beds in Wenchang could induce the production of cellulase with high enzyme activity by SB, further indicating that it will be more instructive for shrimp aquaculture to isolate cellulolytic strains from the environment similar to the culture environment. Generally, the larger the contact area of SB with microorganisms, the more easily the SB can be degraded into available carbon sources by microorganisms, which can promote the growth of microorganisms and improve water quality.
CFW-C18 and MW-M15 showed the excellent SB decomposition capabilities, and the SB decomposition rates of both were up to 63.81% and 48.57%, respectively. Interestingly, MW-M19 with lowest MAR Index and highest SBase also displayed the relatively high SB decomposition rate (up to 29.52%). Overall, CFW-C18 and MW-M19 with relatively high SBase and SB decomposition rate were obtained in this study, which will lay a foundation for the development of cellulase producing marine microbial agents for L. vannamei aquaculture based on the artificial addition of SB as the carbon source.