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Cite this article: Yanagisawa M, Kawamoto N (2018) Isolation and Characterization of Novel Paenibacillus spp. Utilizing Various Carbohydrates Contained in Seaweeds and Exhibiting Growth at Higher Temperatures. JSM Microbiology 6(1): 1047.

*Corresponding authorMitsunori Yanagisawa, Department of Environmental Sciences, School of Food, Agricultural and Environmental Sciences, Miyagi University,2-2-1, Hatatate, Taihaku-ku, Sendai, Miyagi 982-0215, Japan, Tel: 81-22-245-1401; Fax: +81-22-245-1534; Email:

Submitted: 28 September 2017

Accepted: 21 February 2018

Published: 23 February 2018

Copyright© 2018 Yanagisawa et al.

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Keywords•Agar-degrading bacteria•Paenibacillusspp.•Carbohydrates utilization•Maximum growth temperature•Seaweeds

Research Article

Isolation and Characterization of Novel Paenibacillus spp. Utilizing Various Carbohydrates Contained in Seaweeds and Exhibiting Growth at Higher TemperaturesMitsunori Yanagisawa* and Nao KawamotoDepartment of Environmental Sciences, School of Food, Agricultural and Environmental Sciences, Miyagi University, Japan

Abstract

Three agar-degrading bacteria, strains TC-1, TC-2, and TC-3, were isolated and characterized to obtain novel bacteria with the ability to utilize various carbohydrates contained in seaweeds and to grow at temperatures of 40°C and higher. All isolated strains were identified as Paenibacillus spp. by analyzing their 16S rRNA gene sequences. Their abilities to utilize carbohydrates such as glucose, mannitol, agar, alginate, starch, laminarin, and carboxymethyl cellulose (CMC) were investigated. TC-2 and TC-3 utilized all examined carbohydrates excluding CMC, but TC-1 did not utilize mannitol, alginate, or CMC. Although no strains utilized CMC, we found that they could degrade the carbohydrate. All isolated strains probably have the ability to degrade cellulose because of their abilities to degrade CMC, which is a cellulose derivative. In addition, the maximum growth temperatures of these strains were investigated. TC-1 and TC-3 grew at 40°C, which is relatively high compared to the growth temperatures of other agar-degrading bacteria, whereas TC-2 could grow at 45°C. We confirmed that TC-2 and TC-3 can utilize various carbohydrates contained in seaweeds and grow at high temperatures, and these strains will be useful for the bioconversion of seaweeds, such as bioethanol production from seaweeds and composting of seaweed waste.

INTRODUCTIONSeaweeds contain various carbohydrates, such as mannitol,

agar, alginate, starch, laminarin, and cellulose, and thus, microorganisms utilizing these carbohydrates are useful for the bioconversion of seaweeds, such as bioethanol production from seaweeds and composting of seaweed waste. Recently, seaweeds have been considered promising raw materials for bioethanol production [1]. In particular, the conversion of alginate and agar into ethanol has attracted interest, and some researchers have used alginate- and agar-utilizing microorganisms and their genes to produce ethanol from these carbohydrates [2-5]. Sphingomonas sp. A1, an alginate-utilizing bacterium, was transformed to express enzymes for producing ethanol from alginate [2]. A gene encoding an enzyme for alginate degradation derived from Pseudo alteromonas sp. SM0524 and a DNA fragment encoding enzymes for alginate transport and metabolism from Vibrio splendidus 12B01 were used to produce ethanol from alginate [3]. Kim et al. [4], expressed genes encoding agar-degrading enzymes from Saccharophagus degradans 2-40 and Streptomyces coelicolor A3 in Escherichia coli and used the recombinant enzymes from the recombinant E. coli to produce ethanol from agarose, a major component of agar. Genes encoding agar-degrading enzymes

from Cellvibrio sp. OA-2007 were also expressed in E. coli and yeast to produce ethanol from agarose [5].

In addition, microorganisms utilizing carbohydrates present in seaweeds are useful for composting of seaweed waste, such as washed-up seaweeds and inedible parts of seaweeds. Seaweed waste contains a large amount of water, which increases the energy requirement for its incineration disposal. Therefore, composting is an efficient disposal method for seaweed waste, and microorganisms utilizing the carbohydrates contained in seaweeds will enhance the carbohydrate decomposition rate in the composting of seaweed waste. In fact, Tang et al. [6], reported that inoculation with the alginate-degrading bacterium Gracili bacillus sp. A7 enhances alginate degradation in the composting of the brown seaweed Undaria pinnatifida.

Microorganisms that utilize multiple carbohydrates can decompose these carbohydrates during composting, and genetic engineering of these microorganisms to produce ethanol facilitates the conversion of multiple carbohydrates into ethanol.Furthermore, microorganisms that can grow at high temperatures are also promising for such applications. The degradation of polysaccharides is a key step in the ethanol production process, and the enzymatic degradation of polysaccharides, such as


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JSM Microbiology 6(1): 1047 (2018) 2/6

cellulose, agar, andstarch, at high temperatures provides some benefits [7-9]. Enzymatic degradation at high temperatures requires thermostable enzymes, and microorganisms growing at high temperatures have potential as sources of thermostable enzymes. During composting, the temperature of the composting mixture is reported to increase due to self-heating [10], and thus, organic materials are decomposed at high temperatures by microorganisms that can grow at these temperatures.

However, no microorganism utilizing all the aforementioned carbohydrates and growing at temperatures of 40°C or higher has been reported to our knowledge. Ohta et al. [8], succeeded in isolating the agar-degrading bacterium Microbulbifer thermotolerans JAMB-A94, which exhibited growth at 52°C, and cloning a gene encoding a thermostable agar-degrading enzyme from this strain. Further characterization revealed that this strain utilizes mannitol and degrades starch [11], but its ability to utilize laminarin, alginate, and cellulose remains unclear. Conversely, the alginate-degrading bacterium Gracili bacillus sp. A7 has been used in the composting of seaweed as mentioned previously [6]. The temperature of the composting mixture was increased to approximately 50°C in this study. Although this strain may grow at higher temperatures, there is no report regarding its ability to utilize carbohydrates other than alginate. In this study, we obtained novel bacteria with the ability to utilize various carbohydrates contained in seaweeds and to grow at temperatures of 40°C and higher by isolating and characterizing three agar-degrading bacteria.

MATERIALS AND METHODS

Isolation and identification of agar-degrading bacteria

A commercial fertilizer produced from seaweed was pur-chased from a maker in Tottori prefecture, Japan.Agar-degrading bacteria, namely strains TC-1, TC-2, and TC-3, were isolated from the fertilizer as follows. One gram of the fertilizer was added to 9 mL of water and homogenized at 10,000 rpm for 10 min using a homogenizer. The homogenized suspension was spread onto Ma-rine agar medium (Marine Broth 2216 containing 15 g/L agar). Bacterial colonies appeared on the agar medium after incubation at 30°C for 5 days, and some colonies formed pits around them. Pit-forming colonies were isolated as agar-degrading bacteria via repeated streaking and culture on the agar medium. The isolated agar-degrading strains were identified by morphological and 16S rRNA gene sequence analyses at TechnoSuruga Laboratory Co., Ltd. (Shizuoka, Japan).The 16S rRNA genes were amplified by PCR using primer set 9F (5’-GAGTTTGATCCTGGCTCAG-3’) and 1510R (5’-GGCTACCTTGTTACGA-3’). Sequencing of the amplified 16S rRNA genes were performed using primers, 9F, 785F (5’-GGATT-AGATACCCTGGTAGTC-3’), 1099F (5’-GCAACGAGCGCAACCC-3’), 536R (5’-GTATTACCGCGGCTGCTG-3’), 802R (5’-TACCAGGG-TATCTAATCC-3’), and 1510R. Phylogenetic analysis using 16S rRNA gene sequences of the isolated strains and closely related strains was conducted by the neighbor-joining method.

Carbohydrate utilization by strains TC-1, TC-2, and TC-3

The abilities of TC-1, TC-2, and TC-3 to utilize carbohydrates

contained in seaweeds were investigated. Glucose, mannitol, agar, alginate, starch, laminarin, and carboxymethyl cellulose (CMC) were used as carbohydrates. The strains were streaked onto Marine agar medium and cultured at 30°C for 72 h. A colony of each strain on the agar medium was inoculated into 10 mL of the basal medium (yeast extract 2.0 g/L, (NH4)2SO4 1.0 g/L, MgSO4∙7H2O 1.1 g/L, K2HPO4 7.0 g/L, KH2PO4 2.0 g/L, final pH 7.0) containing 1.0 g/L agar and precultured at 30°C and 120 rpm for 48 h. Then, 0.1 mL of the precultured medium was inoculated into 10 mL of the basal medium and seven types of basal medium containing 2.0 g/L carbohydrates, as described previously. The strains were cultured at 30°C and 120 rpm for 72 h, and cell densities were measured using optical density at 600 nm. When the cell density of a strain grown in a carbohydrate-containing medium was higher than that of the strain grown in a carbohydrate-free medium, we judged that the carbohydrate was utilized by the strain.

CMC degradation by strains TC-1, TC-2, and TC-3

The abilities of TC-1, TC-2, and TC-3 to degrade CMC were investigated. Colonies of the strains grown on Marine agar medium were picked and inoculated onto a plate medium (basal medium containing 2.0 g/L glucose, 3.0 g/L CMC, and 15 g/Lgellan gum). The plate medium inoculated with the strains was incubated at 30°C for 7 days. The plate medium was then stained with Congo red stain solution and destained with 1 mol/L sodium chloride. The ability of each strain to degrade CMC was confirmed by the appearance of halos around the colonies, and the ratios of the diameters of the halos to those of the colonies were measured to compare the CMC degradation activities of the strains.

Culture of strains TC-1, TC-2, and TC-3 at different temperatures

The maximum growth temperatures of TC-1, TC-2, and TC-3 were investigated by culturing these strains at different temperatures. The strains were precultured in 10 mL of the basal medium containing 1.0 g/L agar at 30°C and 120 rpm for 48 h. Then, 0.1 mL of the precultured medium was inoculated into 10 mL of the basal medium containing 2.0 g/L glucose and cultured at 120 rpm for 72 h. TC-1 and TC-3 were cultured at three different temperatures (35, 40, and 45°C); TC-2 was cultured at four different temperatures (35, 40, 45, and 50°C). During culture, cell density was measured using optical density at 600 nm every 24 h.

Statistical analysis

Significant differences between the cell densities measured using optical density at 600 nm were evaluated using Dunnett’s test.

RESULTS AND DISCUSSION

Isolation and identification of agar-degrading bacteria

Three agar-degrading bacteria, named strains TC-1, TC-2, and TC-3, were isolated from commercial fertilizer. All strains were rod-shaped bacteria, and the cells of TC-1, TC-2, and TC-3 were 0.8–0.9 × 1.5–3.0, 0.7–0.8 × 1.5–3.0, and 0.8–0.9 × 1.5–2.5 μm




in size, respectively. The nucleotide sequences of the bacteria’s 16S rRNA genes were determined to identify these strains. The 16S rRNA gene sequence analysis and BLAST search illustrated that TC-1 has the highest similarity to Paenibacillus agarexedens DSM1327 (99.0%), and TC-2 and TC-3 have the highest similarities to Paenibacillus nanensis MX2-3 (98.4 and 97.1%, respectively).

A phylogenetic tree based on the 16S rRNA gene sequences of the isolated strains and closely related strains was constructed (Figure 1). TC-1, TC-2, and TC-3 were classified into the Paenibacillus genus and designated as Paenibacillus sp. TC-1, Paenibacillus sp. TC-2, and Paenibacillus sp. TC-3, respectively. The 16S rRNA gene sequences of these strains were deposited in DNA Data Bank of Japan (DDBJ) under accession nos. LC271675–LC271677.

Carbohydrate utilization by TC-1, TC-2, and TC-3

TC-1, TC-2, and TC-3 were cultured in carbohydrate-free and carbohydrate-containing media, and the cell densities of the cultures were measured using optical density at 600 nm (Figure 2). Cell densities of TC-1 after cultivation in glucose-, agar-, starch-, and laminarin-containing media were higher than those after cultivation in carbohydrate-free medium (Figure 2a), indicating that this strain can utilize these carbohydrates for its growth. On the contrary, the cell densities of TC-1 grown in mannitol-, alginate-, and CMC-containing media were similar to that of the strain in carbohydrate-free medium, suggesting that the strain does not use these carbohydrates as substrates (Figure 2a). As brown seaweeds contain high levels of mannitol and alginate, TC-1 does not appear useful for the bioconversion of brown seaweeds.

TC-2 and TC-3 were able to utilize all carbohydrates excluding CMC (Figure 2b and c). Therefore, these strains must possess various genes encoding polysaccharide-degrading and carbohydrate-metabolizing enzymes. The polysaccharide-degrading enzymes obtained from these strains can be used for the degradation of polysaccharides in ethanol production from seaweeds. The expression of genes encoding polysaccharide-degrading enzymes in other microorganisms will produce recombinant enzymes for polysaccharide degradation. Genes encoding carbohydrate-metabolizing enzymes in TC-2 and TC-3 may enable ethanol-producing microorganisms to produce ethanol from non-fermentable carbohydrates in seaweeds. Furthermore, if a method for genetically transforming these strains is developed, ethanol will be directly produced from multiple carbohydrates contained in seaweeds.

None of the isolated strains utilized CMC, which was used to investigate the abilities of the strains to utilize cellulose. CMC is a cellulose derivative, and its degradation product is not glucose, which is the degradation product of cellulose. A strain that does not metabolize the degradation product of CMC cannot utilize CMC even if it has the ability to degrade it. Therefore, it is unclear whether the isolated strains can degrade CMC, leading us to investigate their abilities to degrade it.

CMC degradation by TC-1, TC-2, and TC-3

TC-1, TC-2, and TC-3 were cultured in the plate medium containing CMC, and their abilities to degrade CMC were evaluated by Congo red staining (Figure 3). Halos appeared around the colonies of all strains, and these strains were confirmed to possess CMC-degrading ability. The diameters of the colonies of TC-1, TC-2, and TC-3 were 3.7 ± 1.2, 9.0 ± 1.0, and 3.0 ± 1.0

Figure 1 Phylogenetic tree based on 16S rRNA gene sequences of the isolated strains and the closely related strains.




Figure 2 Cell densities of the strains TC-1 (a), TC-2 (b), and TC-3 (c) after cultivation in different carbohydrates-containing media. The error bars indicate standard deviation for the averaged values (n = 3). The asterisks indicate P< 0.05 versus the cell density in carbohydrate-free medium evaluated using Dunnett’s test.

Figure 3 Detection of the activity of the strains TC-1 (a), TC-2 (b), and TC-3 (c) to degrade CMC.

mm, respectively, whereas the diameters of their halos were 12.7 ± 1.2, 26.3 ± 0.6, and 33.3 ± 1.2 mm, respectively. The ratios of the diameters of the halos to those of the colonies of TC-1, TC-2, and TC-3 were 3.6 ± 0.7, 2.9 ± 0.3, and 11.9 ± 3.8, respectively. Because the ratio of the diameter of the halo to that of the colony probably corresponds to the level of CMC degradation activity, TC-3 has the highest CMC degradation activity among these strains. Conversely, these strains could not utilize CMC (Figure 2), indicating that they degrade CMC but do not metabolize its degradation product. TC-1, TC-2, and TC-3 probably have the ability to degrade cellulose because of their abilities to degrade CMC.

Culture of TC-1, TC-2, and TC-3 at different temperatures

TC-1, TC-2, and TC-3 were cultured in the medium containing glucose as carbohydrateat different temperatures, and the cell densities of their cultures were measured using optical density at 600 nm every 24 h (Figure 4). TC-1 and TC-3 grew at 35 and 40°C (Figure 4a and c). Some agar-degrading bacteria, such as Microbulbifer sp. 6532A [12], Simiduia agarivorans SA1T

[13], Seonamhaeicola algicola Gy8T [14], and M. thermotolerans

JAMB-A94 [8], can grow at temperatures of 40°C or higher, whereas other agar-degrading bacteria do not possess this ability. Therefore, TC-1 and TC-3 are more capable of growth at high temperatures than many other agar-degrading bacteria. These strains did not grow at 45°C (Figure 4a and c), and thus, we did not examine their growth at 50°C. TC-2 grew at 35, 40, and 45°C, whereas it did not grow at 50°C (Figure 4b). We confirmed that the maximum growth temperature of TC-2 is higher than those of TC-1 and TC-3 and many other agar-degrading bacteria.

TC-2 and TC-3 can utilize various carbohydrates contained in seaweeds and grow at higher temperatures. These strains were compared with other polysaccharide-degrading bacteria regarding their abilities to utilize carbohydrates contained in seaweeds and their maximum growth temperatures (Table 1). As glucose is contained in seaweeds as a component of polysaccharides such as starch, laminarin, and cellulose, the ability of the bacteria to utilize glucose was not compared. In addition, bacteria confirmed to degrade a polysaccharide were considered to have the ability to utilize the same polysaccharide in this study. The polysaccharide-degrading bacterium M. thermotolerans JAMB-A94 can grow at 52°C, which was an extremely high growth temperature among the polysaccharide-degrading bacteria examined. Although this strain may utilize all carbohydrates listed in Table 1, its ability to utilize laminarin, alginate, and cellulose has remained unclear as described in the Introduction. S. algicola Gy8T has a maximum growth temperature of 40°C, which is relatively high among polysaccharide-degrading bacteria. However, the abilities of this strain to utilize mannitol and degrade laminarin are unknown. S. agarivorans SA1T and Microbulbifer sp. 6532A can grow at 40°C, but they do not utilize mannitol and laminarin, respectively. Microbulbi fermangrovi DD-13T, Saccharophagus degradans 2-40T, Sabulilitoribacter multivorans M-M16T, and Winogradskyella multivorans T-Y1T,




Table 1: Comparison of the abilities of polysaccharide-degrading bacteria to utilize carbohydrates and their maximum growth temperatures.

CharacteristicStrain*

1 2 3 4 5 6 7 8 9 10

Carbohydrate utilization†

Mannitol + + + ND‡ − ND‡ − ND‡ − −

Agar + + + + + + + + +§ +§

Alginate + + ND‡ + + + + + + +

Starch + + + + + ND‡ + + + +

Laminarin + + ND‡ ND‡ ND‡ − + + +¶ +¶

Cellulose +** +** ND‡ + + + + + +** +**

Maximum growth temperature (°C) 45 40 52 40 40 40 38 37 37 33*1, Paenibacillus sp. TC-2 (present study); 2, Paenibacillus sp. TC-3 (present study); 3, Microbulbifer thermotolerans JAMB-A94 [8,11]; 4, Seonamhaeicola algicola Gy8T[14]; 5,Simiduia agarivorans SA1T[13]; 6, Microbulbifer sp. 6532A [12]; 7, Microbulbifer mangrovi DD-13T[15]; 8, Saccharophagus degradans 2-40T[16]; 9, Sabulilitoribacter multivorans M-M16T[17]; 10, Winogradskyella multivorans T-Y1T[18]†+, positive; −, negative‡ND, not described§Positive for agarose degradation¶Positive for curdlan degradation**Positive for CMC (carboxymethyl cellulose) degradation

degrading enzymes useful for polysaccharide degradation in ethanol production. TC-1 also may possess thermostable enzymes for the degradation of agar, starch, laminarin, and cellulose, as it utilizes these carbohydrates and grows at high temperatures. Furthermore, we anticipate that TC-2 and TC-3 will enhance the decomposition of multiple carbohydrates during composting seaweed waste at high temperatures.

CONCLUSIONA microorganism with the ability to utilize multiple

carbohydrates contained in seaweeds and to grow at high temperatures is useful for the bioconversion of seaweeds. However, microorganisms possessing these properties are extremely rare.In this study, we obtained novel bacteria with the ability to utilize various carbohydrates contained in seaweeds and to grow at temperatures of 40°C and higher by isolating and characterizing three agar-degrading bacteria, strains TC-1, TC-2, and TC-3.All isolated strains were classified into the Paenibacillus genus by analyzing their 16S rRNA gene sequences. Their abilities to utilize carbohydrates contained in seaweeds were investigated. Glucose, mannitol, agar, alginate, starch, laminarin, and CMC were used as carbohydrates.TC-1 did not utilize mannitol, alginate, or CMC. This strain does not appear useful for the bioconversion of brown seaweeds.TC-2 and TC-3 were able to utilize all examined carbohydrates excluding CMC. None of the isolated strains utilized CMC, but we found that they could degrade CMC. The maximum growth temperatures of TC-1 and TC-3 were 40°C, which is relatively high compared to the growth temperatures of other agar-degrading bacteria, and that of TC-2 were 45°C.TC-2 and TC-3 can utilize various carbohydrates contained in seaweeds and grow at higher temperatures. Therefore, thesestrains will increase the efficiency of ethanol production from seaweeds and enhance the decomposition rate of carbohydrates during composting of seaweed waste.

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Figure 4 The courses of cell densities of the strains TC-1 (a), TC-2 (b), and TC-3 (c) during cultivation at 35 (●), 40 (■), 45 (▲), and 50oC (×). The error bars indicate standard deviation for the averaged values (n = 3). The asterisks indicate P< 0.05 versus the cell density at 0 h evaluated using Dunnett’s test.

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Yanagisawa M, Kawamoto N (2018) Isolation and Characterization of Novel Paenibacillus spp. Utilizing Various Carbohydrates Contained in Seaweeds and Ex-hibiting Growth at Higher Temperatures. JSM Microbiology 6(1): 1047.

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