Hyaluronic acid production by Klebsiella pneumoniae strain H15 (OP354286) under different fermentation conditions

doi:10.21203/rs.3.rs-3112641/v1

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Hyaluronic acid production by Klebsiella pneumoniae strain H15 (OP354286) under different fermentation conditions

https://doi.org/10.21203/rs.3.rs-3112641/v1

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published 17 Oct, 2023

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Background

Hyaluronic acid (HA) has gained significant attention due to its unique physical, chemical, and biological properties, making it widely used in various industries. This study aimed to screen bacterial isolates for HA production, characterize favorable fermentation conditions, and evaluate the inhibitory effect of bacterial HA on cancer cell lines.

Results

A total of 108 bacterial isolates from diverse sources were screened for HA production using HPLC, turbidity, and carbazole determination methods. Among the HA-producing isolates, Klebsiella pneumoniae H15 isolated from an animal feces sample, was superior in HA production. The strain was characterized based on morphological, cultural, and VITEK MS methods. Molecular identification using 16S rDNA sequencing and phylogenetic analysis confirmed its identity. Fermentation conditions, including pH, temperature, time, and agitation rate, were figured out to maximize HA production. The basal medium, comprising sucrose (7.0%) as the carbon source and yeast extract + peptone (1.25% each) as nitrogen substrates, favored the highest HA production at pH 8.0, for 30 h, at 30°C, and 180 rpm agitation. The average maximized HA concentration reached 1.5 g L^− 1. Furthermore, bacterial HA exhibited a significant inhibitory effect on three cancer cell lines, with the lowest concentration ranging from 0.98–3.91 µg mL^− 1.

Conclusions

K. pneumoniae H15 isolated from animal feces demonstrated promising potential for HA production. The most favorable fermentation conditions led to a high HA yield. The inhibitory effect of bacterial HA on cancer cell lines highlights its potential therapeutic applications. These findings contribute to the understanding and utilization of HA in various industries and therapeutic applications.

Hyaluronic acid (HA)

fermentation parameters

Klebsiella pneumonia

cytotoxicity

Hyaluronic acid, also called hyaluronan or hyaluronate (HA) is a linear, unbranched biopolymer composed of disaccharide repeats of D-glucuronic acid and N-acetylglucosamine which joined alternately by β-1,3 and β-1,4 glycosidic bonds. The molecular weights of HA are highly variable from different sources ranging from 10⁴ to 10⁷ Da comprising about 2,000–25,000 units [1]. Due to its unique physical, chemical and biological properties such as viscoelasticity, distinctive moisturizing retention, high water-holding capacity, biocompatibility, wound healing, coupled with its lack of immunogenicity and toxicity, HA is widely used in several applications such as drug manufacture, ophthalmology, rheumatology, healthcare, cosmetics, and food industry [2–10]. HA was traditionally extracted from animal tissues, however some limitations were found, such as cross-species viral infection and immunogenicity due to the use of hazardous solvents [11]. Additionally, animal-derived HA is complexed with proteoglycans which is frequently associated with enzymes that degrade HA. Hence, the use of microbial fermentation for HA production with lower production costs and less environmental pollution is an urgent need [12, 13]. HA derived from polymers produced from bacteria was found identical with eukaryotic HA [14, 15]. The HA capsule is a natural virulence factor in immune system of some bacteria such as Streptococcus sp. [16]. HA has been successfully produced in high productivity by Streptococcus spp. as the main producer; notable S. equi, S. equisimilis, S. pyogenes and S. uberis [13, 17]. Nevertheless, the production of HA from Streptococcus spp. is facing a growing concern due to the fact that streptococci are pathogenic [18]. In this regard, HA production with recombinant microorganisms such as Bacillus subtilis, Lactococcus lactis, Escherichia coli, Agrobacterium sp., S. equi subsp. zooepidemicus has attracted a considerable attention [1]. Culture conditions such as pH, temperature, agitation speed, aeration rate, shear stress, dissolved oxygen, and bioreactor type showed great effect on production of microbial metabolites, in general, and significantly influence the microbial HA production, in particular, [1, 19]. In literature, most studies have focused on production of HA by Streptococcus sp. The search for new bacterial species for HA production is an essential demand. Moreover, little is known about the physiology of HA fermentation. From the technical, economic and environmental perspectives, it is desirable to develop high productivity of microbial fermentation of HA [20, 21]. Thus, developing a cost-effective HA fermentation process by microorganisms becomes an essential issue [22–26]. As a starting point in this manuscript, 108 bacterial isolates from various sources were screened for HA production. Nine isolates were positive for HA production and one promising strain of Klebsiella pneumoniae isolated from animal feces was used as attractive source for HA production in this paper. The most favorable fermentation conditions for enhanced HA production by this strain were demonstrated. The feasibility of using HA as antitumor agent was also documented in this paper.

Screening of bacterial strains for the production of HA

Among 108 bacterial isolates recovered from different sources (soil, animal excretions, food and patients' samples), nine isolates (8.33%) were found positive for HA production using a modified liquid medium. The isolates were screened for HA production using HPLC, turbidity and carbazole method. Using the standard HA, a standard curve of turbidity and carbazole method was made to quantify HA in the extracted bacterial supernatants (Fig. 1A and B). R² indicating the functional linear relationship between HA concentration and the absorbance was above 0.986 for carbazole method and above 0.970 for turbidimetric method, as shown in the calibration graphs (Fig. 1A and B). The presence HA in the crude extracts of the bacterial strains was confirmed by HPLC analysis of the HA-positive strains initially identified by VITEK MS, six strains of K. pneumoniae were dominant in HA production with varied potentiality (Table 1). K. pneumoniae H15 (isolated from animal feces sample) was the superior strain in HA production achieving 891, 932 and 940 mg HA L^− 1 using HPLC, turbidity and carbazole method, respectively; followed by S. haemolyticus HB9 (isolated from a patient blood sample) that produced 104, 176 and 232mg HA L^− 1, respectively. B. cereus HW29 and E. coli AU5 showed a low HA-producing ability, compared with the two superior strains. Using HPLC determination, the first strain (HW29 isolated from a patient wound sample) showed a sharp less HA potentiality by 15.63 folds than that of H15 strain. The second strain (AU5 isolated from animal urine) produced HA in an amount less than that of H15 strain by 19.8 folds. The lowest HA-producing ability was obtained by K. pneumoniae CS22 (isolated from a cultivated soil) giving 8.00 mg HA L^− 1(Table 1).The HPLC analysis confirms the identity of HA separated from the bacterial cultures showing a peak with a retention time of 1.28 min that matched the authentic HA under the same conditions (Fig. 1C and D shows identical peak of HA separated from the crude extract of K. pneumoniae H15, the most active isolate with the authentic HA). Therefore, K. pneumoniae H15 was used as experimental organism throughout this study in order to determine some favorable fermentation conditions that may contribute to improving HA production.

Table 1

HA-producing bacterial strains identified by VITEK MS. Calculated mean is for triplicate measurements from two independent experiments ± SD; means with different superscripts in the same column are considered statistically different (LSD test, P ≤ 0.05).
HA-producing bacterial strains	Code no.		Source of isolation	Concentrations of HA (mg L^− 1)
HA-producing bacterial strains	Code no.		Source of isolation	HPLC method	Turbidity method	Carbazole method
K. pneumoniae	CS3	Cultivated soil		90.0 ± 20.0^cd	127 ± 24.0^bc	125 ± 20^bc
K. pneumoniae	CS22	Cultivated soil		8.00 ± 1.00^e	15.0 ± 3.00^e	15.0 ± 1.00^e
K. pneumoniae	AU3	Animal urine		67.0 ± 14.0^cd	92.0 ± 1.00^bc	128 ± 41.0^bc
E. coli	AU5	Animal urine		45.0 ± 15.0^d	85.0 ± 14.0^cd	88.0 ± 11.0^cd
K. pneumoniae	AF17	Animal faces		891 ± 28.0^a	932 ± 20.0^a	940 ± 32.0^a
S. haemolyticus	HB9	Patient blood		104 ± 27.0^cd	176 ± 33.0^cd	232 ± 53.0^bc
K. pneumoniae	HW16	Patient wound		6.00 ± 1.00^e	14.0 ± 2.00^e	13.0 ± 1.00^e
K. pneumoniae	HW24	Patient wound		14.0 ± 1.00^e	45.0 ± 3.00^d	44.0 ± 3.00^d
B. cereus	HW29	Patient wound		57.0 ± 1.00^d	98.0 ± 3.00^cd	102 ± 3.00^cd

Calculated mean is for triplicate measurements from two independent experiments ± SD; means with different superscripts in the same column are considered statistically different (LSD test, P ≤ 0.05).

Morphological and molecular identification of H15 isolate

This isolate H15 was subjected to cultural, morphological and molecular characterization. The cultural characteristics were observed when streaked on BHI gar and blood agar. After incubation at 37ºC for 24h, it appeared as large, opaque, mucoid, and greyish white colonies on BHI agar (Fig. 2A). On blood agar surface, colonies are non-hemolytic with a diameter about 3–4 mm (Fig. 2B). Microscopic examination showed that the isolate is Gram-negative short rod and non-spore forming (Fig. 2C). The H15 isolate was molecularly identified via extraction of the genomic DNA and amplification of rRNA by PCR. The amplicon was then got sequenced and the retrieved sequence was deposited in the GenBank under accession number OP354286. The sequence obtained (1104 bp) was compared against sequences retrieved from GenBank database using BLAST. Using 16S rRNA sequence data, the phylogenetic analysis of the bacterial strain H15 suggested that this strain is closely related to K. pneumoniae. The 1104 bp partial sequence of 16S rRNA was aligned to construct a phylogenetic tree (Fig. 3), and alignment data showed a 98% identity with K. pneumoniae accession numbers MZ389276, and MZ389247, OK178048.

Effect of cultural conditions on the production of HA by K. pneumoniae H15

Effect of initial pH

The production of HA by the strain H15 was analyzed at different pH values of the modified medium using varying amounts of 1 N HCl and 1 N NaOH. Results showed that the final pH values of the culture medium were in between 3.5 and 4.3. The HA production was promoted at the alkaline pH values, and the maximum HA concentrations were significantly obtained at pH 8.0 recording 1517, 1616 and 1575 mg L^− 1 by HPLC, turbidity and carbazole method, respectively (Fig. 4). Considerable HA concentrations of 869, 891 and 913 mg L^− 1 were also obtained by the three respective determinations at the highly alkaline pH 9.0. Comparable HA concentrations were produced by the experimental strain at pH 6.0 and 7.0. Decreasing the pH value to the acidic side (pH5.0) attained sharp decreases in HA production.

Effect of incubation temperature

Data given in Fig. 5 indicated that the HA biosynthesis by the experimental organism was greatly influenced by incubation temperature. Maximum HA production was obtained at 30°C recording 1318, 1376 and 1430 mg L^− 1using HPLC, turbidity and carbazole determination, respectively. At this temperature, significant difference in HA concentrations (P ≤ 0.05) was obtained. Satisfactory concentrations of HA were obtained at 25°C achieving 76.93, 81.61 and 81.12% of HA concentrations produced by the respective three determinations at 30°C. Fermentation conducted at higher temperatures (35 to 45°C) promoted gradual decrease in HA concentrations.

Effect of fermentation time

The effect of time course profile on HA production by the experimental strain was also studied (Table 2). Data obtained showed that HA biosynthesis was increased with increase in the fermentation period till achieving the highest concentration of 1352, 1474 and 1448 mg HA L^− 1 at 30 h of fermentation by HPLC, turbidity and carbazole determination, respectively. After which, the HA concentrations were decreased upon increasing the incubation time at 36 h recording1222, 1266 and 1267 mg HA L^− 1.

Table 2

Effect of fermentation times on HA production by AF17 strain. Calculated mean is for triplicate measurements from two independent experiments ± SD; means with different superscripts in the same column are considered statistically different (LSD test, P ≤ 0.05).
Fermentation time (h)	Concentrations of HA (mg L^− 1)
Fermentation time (h)	HPLC method	Turbidity method	Carbazole method
12	748 ± 58.0^c	791 ± 64.0^c	808 ± 51.0^c
18	860 ± 62.0^c	882 ± 113^bc	905 ± 104^bc
24	903 ± 116^bc	918 ± 171^bc	948 ± 88.0^bc
30	1352 ± 116^a	1474 ± 156^a	1448 ± 115^a
36	1222 ± 68.0^ab	1266 ± 164^ab	1267 ± 91.0^ab

Effect of agitation rate

The production of HA by K. pneumoniae H15 was further analyzed at different agitation rates (Fig. 6). The highest HA concentration was achieved at 180 rpm (the control agitation rate) recording 892, 911 and 933mg L^− 1 using HPLC, turbidity and carbazole determination, respectively. At this agitation rate, significant difference (P ≤ 0.05) in HA concentration was obtained by the three methods of determination. After which, the HA concentration was gradually decreased by increasing the agitation rate. Noteworthy, the agitation at the lowest rate (50 rpm) promoted higher HA concentration than that obtained by the highest agitation rate (250 rpm) using the three HA determinations. At 50 rpm, the HA production was 1.94, 2.36 and 2.31 folds of that obtained at 250 rpm by the three respective determinations.

Effect of C and N sources

In order to investigate the role of nutritional sources on HA production by the experimental strain, various C and N sources were tried and separately added to the basal medium (7.0% and 2.5%, respectively). The highest HA production was attained upon using sucrose (control) as C source achieving 889, 919, and 941 mg HA L^− 1 by HPLC, turbidity and carbazole determination, respectively (Table 3). Fructose and glucose achieved considerable HA production showing insignificant differences with sucrose (P ≥ 0.05). The two respective sources produced 772 − 751, 843 − 785, and 854 − 796 mg HA L^− 1 by the three respective determinations. Lactose and maltose were unsatisfactory C sources for HA production. The decrease in HA concentration produced by lactose was 38.8, 31.8, and 30.5% of that produced by sucrose using the three respective determinations. Using maltose, the decrease in HA concentration was 47.8, 42.2, and 41.1% as compared with sucrose.

Table 3

Effect of C and N sources on HA production by H15 strain.
C and N sources	Concentrations of HA (mg L^− 1)
C and N sources	HPLC method	Turbidity method	Carbazole method
Glucose	751 ± 29.0^a	785 ± 27.0^a	796 ± 64.0^a
Sucrose	889 ± 49.0^a	919 ± 63.0^a	941 ± 93.0^a
Lactose	544 ± 21.0^c	627 ± 30.0^c	654 ± 27.0^c
Maltose	464 ± 41.0^c	531 ± 52.0^c	554 ± 44.0^c
Fructose	772 ± 23.0^a	843 ± 44.0^a	854 ± 52.0^a
Peptone	555 ± 40.0^b	661 ± 29.0^b	683 ± 37.0^b
Yeast	751 ± 34.0^a	861 ± 37.0^a	878 ± 41.0^a
Yeast + peptone	892 ± 36.0^a	924 ± 44.0^a	956 ± 45.0^a
Ammonium sulfate	000 ± 000	000 ± 000	000 ± 000
Sodium Nitrate	000 ± 000	000 ± 000	000 ± 000
Calculated mean is for triplicate measurements from two independent experiments ± SD; means with different superscripts in the same column are considered statistically different (LSD test, P ≤ 0.05).

Upon testing different N sources on HA production by the experimental strain, the highest level was attained upon utilizing yeast extract + peptone (control) as a N substrate achieving 892, 924, and 956 mg HA L^− 1 by the three respective determinations (Table 3). When yeast extract was used as a sole N substrate, the HA concentrations achieved was 751, 861, and 878 mg L^− 1 showing insignificance differences (P ≥ 0.05) with HA concentrations obtained with yeast extract + peptone combination. Upon utilizing peptone as a sole N source, lower HA concentrations were produced which represented 62.2%, 71.5%, and 71.4% of those produced upon utilizing the yeast extract + peptone combination by the three respective determinations. Testing inorganic N substrates showed undetectable HA concentrations produced by the experimental strain. This clearly indicates that organic complex N substrates are essential sources for the biosynthesis of HA.

Cytotoxicity of HA

The cytotoxicity of HA was demonstrated against three cell lines viz. MCF-7 (breast), HepG-2(liver), and HCT(colon) at various concentrations (Table 4, Fig. 7). Data showed that the lowest inhibitory concentration of HA was 0.98 µg mL^− 1 against MCF-7 and HCT cells, whereas, it was 3.91 µg mL^− 1 against HepG-2 cells. The more increase in HA concentration, the more significant decrease in cells proliferation of the cancer cell lines tested. Data further indicated that HCT cells were more sensitive to the cytotoxic activity of HA thanMCF-7 and HepG-2 cells. The estimated IC₅₀value was 273.4 µg mL^− 1 against HCT cells (Table 4). In the case of MCF-7 and HepG-2 cells, the IC₅₀ was 1538 and 1209 µg mL^− 1, respectively (Data not shown). At 500 µg HA mL^− 1 (the higher concentration tested), the cell viability recorded was 57.4% against MCF-7, 63.3% against HepG-2, and 27.8% against HCT.

Table 4

Anticancer activity of HA against three cancer cell lines.
HA conc. (µg mL^− 1)	Cell viability (%)
HA conc. (µg mL^− 1)	MCF-7 (breast)	HepG-2 (liver)	HCT (colon)
500	57.40 ± 1.96^e	63.33 ± 2.41^d	27.83 ± 6.93^d
250	64.69 ± 1.21^d	64.59 ± 1.66^d	58.00 ± 2.63^c
125	74.58 ± 5.21^cd	72.52 ± 1.85^c	75.33 ± 3.74^b
62.5	74.69 ± 7.25^c	73.19 ± 2.11^c	77.83 ± 4.11^b
31.25	76.88 ± 3.96^c	76.67 ± 3.33^bc	77.83 ± 3.62^b
15.63	80.52 ± 5.24^bc	81.85 ± 1.17^b	78.33 ± 2.22^b
7.81	89.69 ± 1.62^b	93.78 ± 2.86^a	79.00 ± 4.62^b
3.91	95.10 ± 2.82^ab	99.63 ± 3.10^a	79.33 ± 3.85^b
1.95	98.33 ± 1.66^a	100 ± 5.23^a	84.17 ± 2.71^b
0.98	99.48 ± 2.17^a	100 ± 3.62^a	98.33 ± 2.46^a
0.49	100 ± 1.29^a	100 ± 4.22^a	100 ± 3.82^a
0.24	100 ± 2.33^a	100 ± 2.63^a	100 ± 6.23^a
IC₅₀(µg mL^− 1)	-	-	273.40
Means ± SD in the same column with different superscriptsare significantly different (LSD test, P ≤ 0.05).

Table 9

Anticancer activity of HA against various cancer cell lines.
HA conc. (µg mL^− 1)	Cell viability (%)
HA conc. (µg mL^− 1)	MCF-7 (Breast)	HepG-2 (Liver)	HCT (Colon)
5000	7.81 ± 1.02^e	37.22 ± 2.33^f	41.86 ± 3.26^d
2500	25.95 ± 1.12^d	45.93 ± 1.47^e	43.04 ± 2.16^d
1250	65.95 ± 2.92^c	46.85 ± 2.81^e	68.63 ± 3.25^c
625	79.76 ± 3.99^b	55.93 ± 3.26^d	80.88 ± 1.25^b
312.5	100 ± 4.12^a	60.93 ± 1.27^d	95.78 ± 4.92^a
156.25	100 ± 5.23^a	70.93 ± 1.86^c	95.88 ± 5.62^a
78.125	100 ± 1.86^a	77.59 ± 1.22^b	100 ± 2.14^a
39.0625	100 ± 2.56^a	79.44 ± 3.52^b	100 ± 3.25^a
19.53125	100 ± 3.17^a	79.81 ± 4.11^b	100 ± 4.17^a
9.765625	100 ± 3.05^a	83.89 ± 2.85^b	100 ± 5.23^a
4.882813	100 ± 2.81^a	86.30 ± 3.92^b	100 ± 2.81^a
2.441406	100 ± 2.63^a	98.89 ± 2.11^a	100 ± 3.66^a
IC₅₀(µg mL^− 1)	1,538.06	1,209.21	1,272.49
^a,b,cMeans in the same column with different superscript letter following them are significantly different (p < 0.05).

Microbial fermentative production of biomolecules gains a continuous growing interest so far worldwide. From the industrial viewpoint, there is a persistent need for production enhancement of low-cost and high-quality biodegradable polymers of microbial origin which are eco-friendly [38]. Among these biopolymers, HA has been immensely used in pharma, medical, biomaterial, and cosmetic applications due to its unique properties such as viscoelasticity, biocompatibility, and water-retention, in addition to its non-immunogenic properties [39]. In particular, no toxic products were generated upon its degradation [40]. Commercially, HA production occurs by two approaches. The first process involves extraction from animal tissues (rooster combs), however some disadvantages are encountered regarding the difficulty in extraction, the quantity loss due to degradation by endogenous hyaluronidase activity, purification is highly expensive and the extracted HA may contain some contaminants such as viruses [41]. The second process is employed from bacterial fermentation (Streptococcus) and the recovered HA is feasible because it is non-immunogenic and biocompatible [42]. The only limitation of this method is the pathogenicity of Streptococcus which is known to produce endotoxins [1]. A solution of this issue was reported via production of HA from cell-free system in order to avoid contamination with endotoxins and reduce the purification costs [18, 43]. Hence, this study has focused on finding new bacterial isolate with positive activity for HA production. The role of some fermentation conditions affecting the HA production process was indicated. In the screening study for HA production by 108 bacterial isolates recovered from soil, animal excretions, food and patients' samples, nine isolates showed their capability for HA production which were initially identified by VITEK MS. These isolates were K. pneumoniae (6 isolates), E. coli (one isolate), S. haemolyticus (one isolate), and B. cereus (one isolate). In this study, HA concentrations were determined by HPLC, turbidity and carbazole methods. Using the HPLC determination, the HA peak of the authentic and that separated from bacterial cultures recorded a retention time of 1.28 min without detection of any interfering peaks. HA quantification by carbazole depends on degradation of HA by sulfuric acid into D-glucuronic acid and N-acetyl glucose amine then carbazole reacts with the produced D-glucuronic acid producing violet color [44]. However, HA determination by carbazole method is laborious and time-consuming due to the usage of sulfuric acid. Hence, the turbidimetric method is a good alternative for carbazole, as this method is very easy and fast and depends on the formation of insoluble complexes between HA and cetyltrimethylammonium bromide (CTAB) that can disappear within 1h [45]. Çağlar [46] applied an HPLC method for determination of HA in pharmaceutical formulations and they found that HA was separated at 7.54 min. In comparison with carbazole and turbidimetric method, HPLC is a better measurement and more accurate, and sensitive method for HA determination with high selectivity [33, 46]. Using HPLC analysis, the HA producing-abilities of flask-cultures were 6.0-891 mg L^− 1 by K. pneumoniae isolates, 45 mg L^− 1 by E. coli isolate, 14 mg L^− 1 by S. haemolyticus, and 57 mg L^− 1 by B. cereus isolate. In literature, HA was mainly produced by Streptococci Group A and C, in particular S. equi and S. zooepidemicus [1, 39]. HA was found to be produced by S. equi subsp. zooepidemicus ATCC35246 (2 g L^− 1 [47], S. zooepidemicus G1(ATCC 39920, 2.43 g L^− 1, [48], S. equi subsp. Zooepidemicus ATCC 35246 (3.5 g L^− 1, [49], and Streptococcus equisimilisCVCC55116 (174.7 mg L^− 1, [50]). Other genetically modified bacterial species such as Bacillus, Agrobacterium, E. coli and Lactococcus were reported [51]. An engineered E. coli strain JM109 was found to produce 1.5 g HA L^− 1. Our data showed that K. pneumoniae H15 was superior in HA production (891 mg L^− 1). We have only quoted a paper reports the HA production by Klebsiellae sp. L-10 NTG 50 in a concentration of 2900 mg L^− 1 and by K. pneumonia strain in concentration of 48 mg L^− 1 [52]. Hence, K. pneumoniae H15 was nominated as a promising isolate for HA production in this study.

The morphological characteristics of K. pneumoniae H15 on BHI gar and blood agar are in agreement with the previous studies [53–55]. In this study, the isolate was identified by VITEK 2 system which is a computerized microbiology program using growth based technology. In the same line, Abbas [55] applied the VITEK 2 system in order to identify pathogenic K. pneumoniae. The identification of our strain was further confirmed based on16S rRNA sequencing carried out by the NCBI Blast service and GenBank. BLAST analysis data based on sequence homology showed that the strain had 98% identity with K. pneumonia strains MZ389276, MZ389247, and OK178048.It was deposited in the GenBank with accession number OP354286. The phylogenetic tree inferred from the 16S rRNA sequence was correlated well with the species that was defined by cultural and morphological characteristics in the universal keys [53, 56].

The current study was directed to demonstrate the effect of some cultural conditions on HA production by K. pneumoniae H15. Upon testing the effect of pH of the medium on HA production by the experimental strain, maximum HA concentrations were attained at pH 8.0 (1517, 1616 and 1575 mg HA L^− 1 by HPLC, turbidity and carbazole method, respectively). It is evident that HA production was favored at the alkaline pH values. In the same line, Pan [57] reported that the highest HA production by Streptococcus zooepidemicus was achieved at pH 8.0. Additionally, Ucm [39] reported that culture medium pH of the initial stage of fermentation should be maintained at 8.0 in order to enhance the production of MW HA. Similarly, Chen (50) attained the highest HA production (174.7 mg L^− 1) by Streptococcus equisimilis mutant NC2168 at pH 7.8. Gedikli [17] set the pH of the culture medium at 7.2 in order to maximize HA production (0.87 g L^− 1) from Streptococcus equi isolate. On the other hand, Johns [58] found that pH 6.7 ± 0.2 is the most favorable for HA production by S.zooepidemicus sub-species zooepidemicus ATCC 35246. Noteworthy, it was reported that the optimum growth of K.pneumoniae strains was attained in a range of pH 6–8 [59].

The production of HA by our bacterial strain was greatly affected by incubation temperature and time. The highest HA levels (1318, 1376 and 1430 mg L^− 1 using HPLC, turbidity and carbazole determination, respectively) were significantly obtained at 30°C. Ucm [39] reported that temperature of the initial stage of fermentation should be maintained at 31°C in order to enhance the production of MW HA. Similarly, Chen [50] found that 33°C was the optimum temperature for HA production by Streptococcus equisimilis mutant NC2168. By contrast, 37°C was reported to be the optimum temperature for HA production by S. equi ATCC 6580 and S.zooepidemicus [57, 60]. Our Data further showed that 30 h of fermentation was the most favorable incubation time for HA production by K. pneumoniae H15. At this time, maximum HA levels (1352, 1474 and 1448 mg L^− 1) were obtained by HPLC, turbidity and carbazole method, respectively. Reports on the favorable incubation time for bacterial production of HA are varied. Gedikli [17] stated that 24 h was the optimum incubation time for HA production by an isolate of S. equi. Kim [60] studied time course of HA production by S. equi ATCC 6580. They found that the molecular weight of HA was accumulated in the culture at 5 h and increased after 15 h of incubation. The authors concluded that the molecular weight of HA production was related to the age of cell growth. Agitation speed was found to be critical factor in the HA production process [17, 58, 60]. The present study found that agitation speed at 180 rpm was the most favorable for HA production by K. pneumoniae H15. At this speed, HA was produced in levels of 892, 911 and 933 mg L^− 1 using HPLC, turbidity and carbazole determination, respectively. In previous studies, the optimal agitation speed for microbial production of HA was varied. The agitation speed at 170 rpm was found the most favorable for HA production [16, 61]. On the other hand, 200 rpm was found the optimal [50, 62–64]. Gedikli [17] found that 250 rpm was the optimum for HA production by S. equi; Johns [58] stated that 600 rpm was the optimum speed for HA production by S. equi sub-species zooepidemicus ATCC 35246; Kim [60] found the optimum speed 1200 rpm for HA production by S. equi ATCC 658.The aerated culture gave higher HA concentration and yield than the equivalent anaerobic fermentation [58]. For example, Armstrong and Johns [65] found a 20% increase in HA concentration when S. zooepidemicus was grown under aerobic conditions. However, it was observed that HA production was not affected by aeration rate, whereas it decreased with the increase of agitation speed [60]. The decrease in the rate of HA synthesis at low agitation rate was counterbalanced by an increased rate of lactate synthesis from glucose [58]. Very high agitation speeds may be deleterious to HA quality, because a high shear rate causes damaging of the HA polymer [58]. Additionally, Hasegawa [66] reported that HA production increased with the increase of aeration rate and agitation speed; moreover, too high agitation speed caused cell damage and led to a drop in HA concentration. Sugar as carbon source is necessary for the glycolytic pathway; it has a critical role in providing energy and used as a precursor for HA biosynthesis [17]. In this study, analysis of HA production at different C sources showed that sucrose (7.0%, w/v) supported the highest HA concentrations (889, 919, and 941 mg L^− 1using HPLC, turbidity and carbazole determinations, respectively). Fructose and glucose gave satisfactory HA concentrations. In the same line, Gedikli [17] found that sucrose (7.0%) afforded the highest HA production (0.45 g L^− 1) by S. equi. Additionally, Pan [57] showed that sucrose followed by glucose were the best C sources for HA production (0.488 and 0.429 g HA L^− 1, respectively) by S. zooepidemicus ATCC 39920. Upon using sucrose as C source in the fermentation medium, HA molecular weight increases [67]. Sucrose was used in the fermentation medium as a C source for HA production [13, 68]. On the other hand, glucose was found as a best C source for HA production by several authors [50, 69, 70]. The effect of C source on HA production may be attributed to the exposure of the organism to stress condition, as a result it produces HA capsule as a barrier against acidic or alkaline pH of the medium [71]. In this study, it was evident that yeast extract–peptone mixture (control of the basal medium) was the most favorable N source for HA production compared with other sole organic and inorganic sources. The combined mixture of yeast extract–peptone produced HA in concentrations of 892, 924, and 956 mg L^− 1 by the three determinations. The same combined mixture of N substrate was recommended for HA biosynthesis by Güngör (13). Additionally, the data obtained in this study indicated that yeast extract as a sole N substrate produced satisfactory HA concentrations. Similarly, Gedikli [17] reported that yeast extract–casein peptone mixture was the most effective N source in HA production yielding 0.5 g HA L^− 1 by S. equi. Pan [57] stated that the more increase in yeast extract concentration, the more increase in HA production. Khue and Vo [72] showed that yeast extract was found to be the best N source for HA production among different sources. In this study, the cytotoxic effect of bacterial HA from K. pneumoniae H15 cultures was tested using MTT assay against three cancer cells (MCF-7, HepG-2, and HCT). Our data showed that the cytotoxic effect of HA was dose dependent where the more increase in HA dose, the more significant decrease in cells proliferation of the cancer cell lines. This was supported by Gedikli [17] who reported on the cytotoxicity of HA and found that the least cytotoxicity was seen on THP-1 cells, the most cytotoxic effect was seen on HUVEC cells. These authors further found that 3 and 6 mg mL^− 1 of HA were statistically cytotoxic on HaCaT and L929 cells and low concentration (1.5 mg mL^− 1) of HA was not cytotoxic on HaCaT and L929 cells. Our results indicated the IC₅₀of HA against HCT, MCF-7 and HepG-2 cells was 273.4, 1538 and 1209 µg mL^− 1, respectively. The difference in IC50 values is dependent on the rate of cell death that might differ due to various factors including HA concentration, incubation time, and cell specificity. HA cytotoxicity, except to origin, depends on molecular weight or concentration of HA. In the literature, some studies demonstrated that high MW HA stimulates cell division; however, some of them cytotoxic [73]. High MW HA inhibits U937 macrophage cell population and induced apoptosis [74]. Harvima [75] claimed that rooster comb and bacterial HA inhibited keratinocyte adherence, at the same time, HA from human umbilical cord stimulate keratinocyte.

Overall, this study highlights characterization of the most favorable cultural conditions for HA production by a promising isolate, K. pneumoniae H15 recovered from animal feces. The findings contribute to expanding the knowledge on HA production using microbial fermentation and provide valuable insights for industrial applications of HA in pharmaceutical, medical, biomaterial, and cosmetic fields. Further research and scale-up studies are warranted to explore the commercial potential of K. pneumoniae H15 for HA production and its downstream applications in various industries.

Samples collection

A total of 108 samples from different sources were collected during April to December, 2020. The samples included 30 samples from cultivated soil with lettuce (at 15 cm depth), 23 samples from animals excretions (10 samples from fresh feces after disinfecting the anal area with 70% ethanol and 13 samples from urine), 25 samples from different foodstuffs collected from shops in Zagazig, Egypt (beef burger, vegetable salad, chicken pane and Shawarma) and 30 samples were collected from patients in Zagazig University Hospital (15 samples from patients' blood and 15 samples from patients' wounds contaminated with pus). All samples were collected in sterile plastic bags and were immediately transferred aseptically in ice box to Bacteriology Lab for isolation.

Bacterial isolation

The bacterial isolates were recovered from the samples collected by serial dilution technique, where one gram of sample (soil, food and feces) was separately suspended in 9-mL of sterile saline (8.5 g NaCl in 1000 mL distilled water) and vortexed vigorously for 15 min to obtain suspension. Each suspension was serially diluted from 10^− 1 to 10^− 9 [27]. Spread plate method was used to isolate the bacteria form the diluted samples where 0.1 ml from each dilution was pipetted out onto plates with nutrient agar and spread with a sterile cotton swab. A loop of urine and blood samples and wound swabs were directly streaked onto nutrient agar plates [28]. All plates were incubated at 37°C for 24 h and the developed bacterial colonies were purified on BHI agar plates using streaking method for getting a pure single colony which were then preserved onto BHI slants after incubation at 37°C for 24 h. Slant cultures were then maintained at 4°C.

Screening of bacterial isolates for HA production

Basal medium used in HA production

A total of 108 bacterial isolates were screened for HA producing ability by cultivation on medium recommended by Güngör [13] which composed of (g L^− 1): sucrose (80 g), peptone (10 g), yeast extract (28 g), K₂SO₄ (0.5 g), MgSO₄ (1.25 g), FeSO₄ (0.008 g), NaCl (1.23 g), and trace elements solution (26 mL composed of CaCl₂, 2 g; ZnCl₂, 0.046 g; CuSO₄.5H₂O 0.019 g per liter), pH adjusted to 7 by using 5M NaOH and HCl before sterilization. A 50 mL of the medium prepared was dispensed in 250 mL Erlenmeyer flasks and autoclaved at 121°C and 1.5 atm for 15–20 min.

Inoculum preparation and cultivation conditions

Trypticasein soy broth (TSB, Lanxess Co, Germany) was used as the seed culture by separately inoculating sterilized test tubes containing 5-mL TSB by bacterial isolates followed by incubation at 37°C for 24 under agitated conditions (125 rpm). One mL of the inoculum prepared was inoculated in the Erlenmeyer flasks containing the production medium which were then incubated at 37°C for 24 h at 180 rpm.

Extraction and separation of HA from bacterial cultures

HA extraction was performed according to the method described by Brown [29], in which, 50 µL of 10% sodium dodecyl sulfate solution (SDS, 100 g in 1 L) was separately added to bacterial culture followed by incubation at room temperature for 10–15 min with shaking for 2 min then 2 mL of 10% cetyltrimethylammonium bromide (CTAB, 100 g in 1 L) was added to Erlenmeyer flasks which were incubated at 10°C for 1 h followed by centrifugation at 4000 rpm for 20 min. The pellets were solubilized in 10 mL of 2M CaCl₂, left for 6 h at 4°C, centrifuged at 4000 rpm for 20 min, and the supernatant was taken and 2 volumes of absolute ethanol was added followed by incubation for 1h at 4°C. After which, centrifugation was employed at 4000 rpm for 20 min and the pellet was solubilized in 10 mL bi-distilled water for overnight at 4°C with shaking in order to dissolve the pellet. Centrifugation was performed at 4000 rpm for 20 min and the supernatant was collected and equal volume of 1% NaCl was added followed by extraction with 2 volumes of absolute ethanol. The solution was centrifuged at 4000 rpm for 20 min and the pellet was then solubilized in 5 mL bi-distilled water for overnight at 4°C. The step of centrifugation and solubilization of the pellet was repeated and the resultant supernatant was filtered with nitrocellulose mixed ester syringe filters (0.45 µ) and the extracted sample was stored at 4°C for further analysis.

Quantification of HA

Carbazole method

Carbazole-sulfuric acid method was used as a calorimetric determination based on Cesaretti [30]. Two reagents were prepared in this method; reagent1 (4.77 g disodium tetraborate in 500 ml sulfuric acid) and reagent 2 (0.125 g carbazole in 100 mL absolute methanol). Sample (1 mL) in test tube was placed in ice water and cooled to 4°C. Freshly prepared reagent 1 (5 mL) was added to the test tube which was closed with caps, shaken and placed in boiling water bath (95–100°C) for 15 min then cooled at room temperature. Reagent 2 (0.2 mL) was added to the test tube and put again in a boiling water bath (95–100°C) for 15 min. After this, they were cooled to room temperature and transferred to Enzyme Linked Immunosorbent Assay (ELISA) plate and the absorbance (color) of the solutions was read at wavelength 530 nm at visible light spectrophotometer against the blank sample in plate reader [31]. The average optical density sample reading was calculated and the HA concentration was determined from a standard curve.

Turbidity method

Two reagents were prepared in this method of HA determination based on Oueslati [32]; reagent 1 (2 g NaOH in 100 mL distilled water) and reagent 2 (2.5 g CTAB in 100 mL reagent 1). Sample (1 mL) was added in test tube and incubated at 37°C for 15 min then 2 mL of reagent 2 was added followed by incubation at 37°C for 10 min and shaking for 10 S at the beginning and at the end of this incubation. The sample was transferred to ELISA plate and the absorbance (turbidity) of the solutions was read at 600 nm against the blank sample in plate reader. The average optical density for each reading of samples was calculated and the HA concentration in the sample was determined from HA standard curve [32].

HPLC method

A chromatography (Young Lin 9100 instrument, YL 9100, Anyang, South Korea) coupled with UV Spectrophotometric detector and equipped with a vacuum degasser (YL9101), a quaternary pump (YL9110), a column thermostat (YL9131), a diode array detector (YL9160), an auto sampler (YL9150), and software Clarity (version 7.3, Data Apex) was used for all measurements. Chromatographic separations of HA were done by a Great Smart RP18 Aq (150 × 4.6 mm, 3 µm) column (Columbia, USA). The column C18 was tempered at 35°C. The packing injection volume was 5 µL. The mobile phase consisted of H₂O: ACN (96:4) (V/V) is freshly prepared and degassed with ultrasound for 30 min and kept in a tightly closed bottle [33]. The freeze-dried HA standard with MW of 1500–1750 KDa was obtained from (Sigma-Aldrich) was used to prepare HA standard by dissolving 0.5 g of HA in 1000 mL distilled water. The solutions were kept in refrigerator at 4°C overnight to obtain a homogenous solution. The Flow rate of mobile phase was adjusted to 0.7 mL min^− 1. The diode array detector was operated at wavelength 205 nm. Chromatograms were evaluated according to retention times and UV spectra of standard and samples [33].

Morphological and cultural characterization of HA-positive isolates

The superior bacterial isolate for HA production was subjected to Gram staining. Its cultural growth was observed on BHI agar and blood agar plates by streaking of loopful of isolate culture (24h-old) on agar plates of the two medium and incubation at 37 ˚C for 24 h.

VITEK MS identification

Positive bacterial isolates for HA production were identified by using Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). A portion of pure colony taken from fresh cultures (24 h-old) was spread on the MALDI-TOF MS slide [34]. The bacterial smear was mixed with 1 mL of the matrix solution (an aromatic organic acid consisted of cyano-4-hydroxycinnamic acid in 2.5% trifluoroacetic acid and 50% acetonitrile) and the target slide was left for 5 min at room temperature until dryness and co-crystallization before placing the slide for further analysis. After that, the sample and the matrix, were exposed to a laser beam to be ionized. The sample's analytes are removed and ionized by the laser beam. The produced peptide mass fingerprints (PMF) were processed by the engine of the computer and then the spectrum classifier algorithm identifies the bacteria automatically by comparing the PMF of the tested organism with the PMF included in the reference database spectra.

Molecular identification of the HA most active isolate

DNA Extraction

The genomic DNA of the most potent HA producing isolates was carried out in Animal Health Research Institute, Giza, Egypt by using the QIAamp DNA Mini Kit. The process of DNA extraction and PCR amplification was carried out according to the manufacturer's instructions.

Primers and PCR Conditions

Prior to sequencing, the rRNA gene was amplified using PCR technique in which two universal primers were incorporated in the reaction mixture. The primers used for gene amplification have the following composition: F27 (AGAGTTTGATCMTGGCTCAG) and R1492 (TACGGYTACCTTGTTACGACTT). 25 µL reaction mixture was prepared in a 0.5 mL PCR tubes as follows: 12.5 µL Emerald Amp GT PCR master mix (2x premix), 4.5 µL PCR grade water, 1 µL Forward primer, 1 µL Reverse primer, 6 µL Template DNA. The PCR amplification was carried out with adjusting the cycling conditions of the primers: one cycle primary denaturation at 94˚C for 5 min; 35 cycles with a 30 sec secondary denaturation at 94 ˚C for 30 secs, annealing at 56˚C for 60 sec and extension at 72˚C for 60 sec. The purified PCR products (amplicons) were reconfirmed using a size nucleotide marker (100–1500 bp) by electrophoresis on 1% agarose gel. These bands were then eluted by using QIAquick gel purification kit. (Qiagen Inc. Valencia CA) and sequenced by using a ready reaction Bigdye Terminator V3.1 cycle sequencing kit (Perkin-Elmer/Applied Biosystems, Foster City, CA), with Cat. No. 4336817. Sequence homologous was identified in the National Center for Biotechnology Information (NCBI) and GenBank database using Basic Local Alignment Search Tool (BLAST) programs [35]. Multiple alignments and phylogenetic interpretations were made using CLC Bio Sequence Viewer (version 7.7, Qiagen Aarhus, Denmark). The sequence (1485 bp) was deposited in the GenBank with accession number (OP354286).

Effect of Cultural Conditions for HA Production

Effect of physical Parameters on HA Production

Effect of various physical parameters on HA production such as incubation temperature (25–45°C), pH (5–9), fermentation period (12–36 h), and different agitation speed (50–250 rpm) were studied by using HA production medium recommended by Gedikli [17].

Effect of nutritional Parameters on HA Production

Several C sources were used to replace sucrose in the basal medium used in production of HA with one of the C sources (glucose, lactose, maltose and fructose) at concentration 70 gm/L and different nitrogen sources such as peptone, yeast, yeast & peptone, ammonium sulfate and sodium nitrate with concentration 25 g L^− 1 were also tested.

Cytotoxicity Effects of HA

Preparation of Different Concentrations of HA

A stock solution of HA separated from K. pneumoniae H15 cultures (10 mg HA were separated from the bacterial cultures, as described earlier in the section of extraction and separation of HA) was prepared. Different concentrations of HA solution were adjusted, as follows: 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, and 0.24 µg mL^− 1 via dilution with distilled water in order to investigate their cytotoxic activities against cell lines in vitro.

Cell Culture and Maintenance

Cell lines viz. breast cancer cell line (MCF-7), human liver cancer cell line (HepG-2) and colon cancer cell line (HCT) were obtained from the American Type Culture Collection (ACC, Rockville, MD, USA), cultured in Dulbecco’s modified Eagle’s medium (DMEM) accompanied by necessary growth factors. The subculture was performed after reaching 90% confluence. The growth media were renewed every day.

Measurement of Cytotoxicity Test (MTT Assay)

Cell suspension was plated in tissue culture (TC) plate for 24 h growth until cells confluence by 90%. For cytotoxicity assay, the cells were seeded in 96-well microtiter plate at a cell concentration of 1×10⁴ cells per well in 100µl of growth medium at 37°C for 24h. Discard culture media from the plate and replaced with a fresh medium. Add serial dilution of HA (12-fold dilution), leave 2 or 3 rows as cell control. Incubate the plate at 37°C for appropriate time (24, 48 and 72 h) in a humidified 5% CO₂ atmosphere. Inspect the plate daily under microscope for detection of morphological changes. Discard media and wash the plate 2times by 200µl washing buffer 7.2 ± 0.2. Add 50 µl / well of MTT solution. Incubate the plate for 3–4 h. at 37°C. Dissolve the intra cytoplasmic MTT formazan crystals using 50 µl DMSO or 0.4% acidic isopropanol/well. Shake the plate for 30 min on plate shaker. Read plate using ELISA reader at 570 nm [36, 37].

Ethics approval and consent to participate

Regarding the research conducted in this study, it is important to note that no animal experimentation was involved. Therefore, animal ethics approval was not applicable to this research.

Consent for publication

Not applicable.

Availability of data and materials

The datasets utilized and examined in this study can be obtained from the corresponding author upon a reasonable request

Competing interests

The authors declare that they have no competing interests.

Funding

This research did not receive any fund. Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Authors' contributions

GE. and A.A. designed and supervised all steps in this study, in addition, revised the final manuscript. S.S. collected samples, performed all microbiological assays, and identified the strains. R.M.A. conducted the tests with data analysis of the results, gave a point of view to discussion, and wrote the draft with the final manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Authors' information

Botany and Microbiology Department, Faculty of Science, Zagazig University, Zagazig 44519, Egypt.

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No competing interests reported.

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Journal Publication

published 17 Oct, 2023

Read the published version in BMC Microbiology →

Editorial decision: Major revision
06 Jul, 2023
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04 Jul, 2023
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Hyaluronic acid production by Klebsiella pneumoniae strain H15 (OP354286) under different fermentation conditions

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

Screening of bacterial strains for the production of HA

Morphological and molecular identification of H15 isolate

Effect of initial pH

Effect of incubation temperature

Effect of fermentation time

Effect of agitation rate

Effect of C and N sources

Cytotoxicity of HA

Discussion

Conclusions

Materials and methods

Samples collection

Bacterial isolation

Screening of bacterial isolates for HA production

Basal medium used in HA production

Inoculum preparation and cultivation conditions

Extraction and separation of HA from bacterial cultures

Quantification of HA

Carbazole method

Turbidity method

HPLC method

Morphological and cultural characterization of HA-positive isolates

VITEK MS identification

Molecular identification of the HA most active isolate

DNA Extraction

Primers and PCR Conditions

Effect of Cultural Conditions for HA Production

Effect of physical Parameters on HA Production

Effect of nutritional Parameters on HA Production

Cytotoxicity Effects of HA

Preparation of Different Concentrations of HA

Cell Culture and Maintenance

Measurement of Cytotoxicity Test (MTT Assay)

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1