Appl Microbiol Biotechnol DOI 10.1007/s00253-013-5296-9
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Isolation and characterization of an efficient bacterial cellulose producer strain in agitated culture: Gluconacetobacter hansenii P2A Yasar Andelib Aydın & Nuran Deveci Aksoy
Received: 8 July 2013 / Revised: 10 September 2013 / Accepted: 26 September 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract In this study, typical niches of acetic acid bacteria were screened for isolation of cellulose producer strains. Hestrin Schramm broth was used as enrichment and production media. Only nine out of 329 isolates formed thick biofilms on liquid surface and were identified as potential cellulose producers. Physiological and biochemical tests proved that all cellulose producers belonged to Gluconacetobacter genus. Most productive and mutation-resistant strain was subjected to 16S rRNA sequence analysis and identified as Gluconacetobacter hansenii P2A due to 99.8 % sequence similarity. X-ray diffraction analysis proved that the biofilm conformed to Cellulose I crystal structure, rich in Iα mass fraction. Static cultivation of G. hansenii P2A in HS medium resulted with 1.89±0.08 g/l of bacterial cellulose production corresponding to 12.0±0.3 % yield in terms of substrate consumption. Shaking and agitation at 120 rpm aided in enhancement of the amount and yield of produced cellulose. Productivity and yield reached up to 3.25± 0.11 g/l and 17.20±0.14 % in agitated culture while a slight decrease from 78.7 % to 77.3 % was observed in the crystallinity index. Keywords Bacterial cellulose . Gluconacetobacterhansenii . 16S rRNA sequence analysis . Mutation
Introduction Bacterial cellulose is the network of cellulose fibrils secreted by certain strains of bacteria, belonging mostly to the Gluconacetobacter genus of Acetobacteraceae family (Vandamme et al. 1998). The members of this family are Y. A. Aydın (*) : N. D. Aksoy Chemical Engineering Department, Istanbul Technical University, 34469, Sarıyer Istanbul, Turkey e-mail: [email protected]
obligate aerobes that have the ability to convert ethanol to acetic acid and, as a consequence, can grow at low pH levels (Kersters et al. 2006; Hwan et al. 2004). Gluconacetobacter sp. can be isolated from a variety of sugary, acidic, and alcoholic environments such as fruits, flowers, vinegar, Kombucha, beer, tequila, and wine (Gossele et al. 1984; Toyosaki et al. 1995; Lisdiyanti et al. 2001; Dellaglio et al. 2005; Nguyen et al. 2010). It is accepted that the cellulosic biofilm produced by these bacteria functions not only by positioning the cells at the surface, where oxygen tension is high, but also by protecting them from the harmful effects of ultraviolet and enemies, yet with a continuous supply of nutrients through diffusion (Iguchi et al. 2000). More recently, Kanchanarach et al. (2010) related polysaccharide formation with acetic acid resistance and proved that the extracellular biofilm produced by rough colonies of Acetobacter pasteurianus strains (IFO3283, SKU1108, MSU10) served for the hindrance of acetic acid diffusion to the cytoplasmic membrane. In addition to its biological function in the survival of microorganisms, bacterial cellulose offers a vast variety of end uses due to its unique material properties. While plant cellulose contains impurities such as lignin, pectin, hemicellulose, and other metabolic products, bacterial cellulose is completely pure and highly crystalline (Raspor and Goranovic 2008). Bacterial cellulose is also renown with its very high water absorptivity reaching up to 200 times its weight (Krystynowicz et al. 2002) and superior mechanical stability with respect to plant-derived cellulose fibers (Shoda and Sugano 2005). The fiber dimensions are also significantly lower for bacterial cellulose, practically in the order of a few nanometers. These special properties have led to the use of this material in a diversity of applications especially in medicine, health, and materials sciences. Several applications were suggested commercially and scientifically of which artificial blood vessels (Klemm et al. 2011), skin and tissue
Appl Microbiol Biotechnol
replacements, acoustic diaphragms (Czaja et al. 2006), electronic displays (Shah and Brown, Jr. 2005), and reinforcement agent in paper and nanocomposites (Siró and Plackett 2010; Chen et al. 2010) are a few. Despite these high-tech end uses, bacterial cellulose production is yet limited by a series of factors affecting process economics (Moon et al. 2006). Conventional method of bacterial cellulose production relies on the static cultivation of bacteria, which is an inefficient, time-consuming, and labor-intensive production scheme (Bae and Shoda 2005). The production capacity of static cultivation is usually very low due to space requirements and insufficient supply of oxygen (Kouda et al. 1997). However, this scheme might be advantageous when pellicle-formed cellulose is required as such in skin replacements or a relatively higher degree of crystallinity is preferable (Raspor and Goranovic 2008). Although enhanced oxygen transfer occurring in agitated fermentation should theoretically increase the rate of cellulose production, most often reduced yields were reported (Shoda and Sugano 2005). The shear stress exerted on the bacterial cells causes spontaneous mutation of Gluconacetobacters by deactivating essential enzymes involved in cellulose synthesis, i.e., phosphoglucomutase and uridine diphosphoglucose pyrophosphorylase (Kornmann et al. 2003; Nguyen et al. 2010). Mutant strains may also show an enhanced ability for accumulation of water-soluble polysaccharides such as acetan (Ross et al. 1991), levan (Bae and Shoda 2005), and xylan (Moon et al. 2006) that use the same starter molecule with bacterial cellulose, i.e., uridine diphosphate glucose, leading also to reduced cellulose production rates. Strategies applied for inhibition of mutation or lowering its effects include addition of supplements such as ethanol (Park et al. 2003a) and agar (Bae et al. 2004), selection of highly productive mutation-resistant strains (Toyosaki et al. 1995), generation of non-acetan (Ishida et al. 2002) or non-ketogluconate (Vandamme et al. 1998) producing mutants, and development of cultivation strategies lowering shear, such as using different impeller types (Kouda et al. 1997), air lift reactors, and rotating disc type reactors (Krystynowicz et al. 2002; Shoda and Sugano 2005). In the current study, we aimed to isolate bacteria possessing high and sustainable ability to produce cellulose from typical niches of acetic acid bacteria. Isolates were characterized in genus level by biochemical and morphological tests. The strain that exhibited highest stability during long-term preservation was subjected to 16S rRNA sequence analysis and characterized in species level. Cellulose production was tested under static, shaken, and agitated conditions. Morphological and structural changes in the synthesized bacterial cellulose were investigated by X-ray diffraction, Fourier transform infrared (FTIR), scanning electron microscopy (SEM), and thermogravimetric analysis (TGA).
Materials and methods Isolation and characterization of microorganisms All chemicals and reagents were either of analytical or biochemical grade. While vinegar fermentation waste was kindly provided by Kemal Kukrer A.S., Turkey, vinegar samples, and fresh and rotten fruits (apples, grapes, apricots, pineapples, chestnuts, and plums) were collected from local markets. Samples were incubated at 28 °C in Hestrin–Schramm (HS) medium (2.0 % D -glucose, 0.5 % peptone, 0.5 % yeast extract, 0.27 % Na2HPO4, and 0.115 % citric acid) for 7 days (Schramm and Hestrin 1954). HS medium was supplemented with 50 mg/l cycloheximide for prevention of the growth of undesired microorganisms such as yeasts. Enriched samples of 0.1 ml volume were streaked onto HS agar (1.5 % agar) plates. Growth was monitored during 3 days of incubation at 28 °C. With repeated streaking, white- to cream-colored mucous colonies were isolated. Isolates were verified as acetic acid bacteria with visualization of CaCO3 clear zones in plates containing CaCO3–ethanol medium (0.05 % D -glucose, 0.3 % peptone, 0.5 % yeast extract, 1.5 % CaCO3, 1.2 % agar, and 1.5 % ethanol). Gluconacetobacter sp. and Acetobacter sp. were further selected by a positive result in overoxidation of acetic acid test as proposed by Dellaglio et al. (2005). Formation of a biofilm at the air vicinity of culture broth after a week of static incubation at 30 °C was accepted as an indication of cellulose production. The pellicle, either stagnant or fragile, was removed by centrifugation, washed with distilled water, retained in 0.1 M NaOH for 20 min at 80 °C, and finally neutralized in 0.1 M acetic acid. After extensive washing with distilled water, any remaining material was either vacuum dried at 0.1 bar and 40 °C or freeze dried at −50 °C and 0.04 mbar. Resistance of the biofilm to this treatment proved conformity to cellulose structure. Strains with cellulose production ability were subjected to further biochemical testing according to the methods described by Asai (1958), Yamada et al. (1976), and Lidiyanti et al. (2001). Gluconacetobacter xylinus LMG 18788 and Gluconacetobacter hansenii DSM 5602 were used as reference strains for biochemical testing. All isolates and reference strains were preserved by freezing in glycerol at −20 °C and monthly checked for strain stability. Sequencing of 16S rRNA gene Most productive strain was identified by 16S rRNA sequencing. Accordingly, the 16S rRNA gene was amplified by PCR with 20F and 1492R primers. The purified PCR products were sequenced directly by 27F, 357F, 530F, 343R, 907R, 1100R, 1392R, and 1492R universal primers using Beckman Coulter sequence analysis tool (CEQ™ DTCS Quick Start Kit, Beckman Coulter). A BLAST search of the determined gene sequence provided highly similar sequences from the same
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phylogenetic group, which were aligned in CLUSTAL Omega (Goujon et al. 2010). A phylogenetic tree was constructed by neighbor-joining method using Geneious 5.6.6 software. The 16S rRNA gene sequence was deposited in GenBank database with the accession number KF155166. The isolate was lyophilized and deposited in Centre for Research and Application of Culture Collections of Microorganisms (KUKENS), Turkey with the reference number KUEN 1606. Culture conditions Stock cultures (1 ml) were grown in 100 ml HS medium at 30 °C under static conditions for 48 h. Vigorous shaking was applied for detachment of bacterial cells and supernatant of this broth was used as seed for batch cellulose production experiments. Inoculation ratio, pH, and cultivation temperature were 10 %, 5.0, and 28 °C, respectively. Static and shaken flask experiments were conducted in 250-ml flasks with 50 ml culture broth, while a 1.5-l jar fermenter (Electrolab, FerMac 200, 1 l working volume) equipped with pH, temperature, and DO (dissolved oxygen) control was used for agitated culture conditions. The pH adjustment was realized by automatic addition of 1 N NaOH and 1 N HCl. The filtered oxygen was supplied at 0.5 vvm. Cultivation periods were a week, 5 days, and 3 days for static, shaken, and agitated conditions, respectively. Samples were taken at definite time intervals to analyze cell growth, cellulose production, and glucose consumption. DNS method was used for glucose assays (Miller 1959). Cell growth was monitored on HS agar plates where any cellulose negative mutants were easily detected and counted by morphological differences. Mutation resistance was tested in five consecutive batches at shaking rates of 120, 150, and 200 rpm in which previous batches were used as inoculants. All experiments were performed in triplicates to ensure the accuracy of the results. Analysis of properties of bacterial cellulose Crystal structure of bacterial cellulose samples were analyzed between 2θ =5–40° at 0.002° angular intervals using an X-ray diffractometer (PANalytical, XPERT PRO). Relative crystallinity index was calculated according to the formula constructed by Segal (1959): CrI ¼
I002 −Iam I002
100
ð1Þ
where CrI is the relative crystallinity index, I002 is the diffraction intensity of 002 plane, and Iam is the intensity of the peak at 2θ =18°, corresponding to crystal and amorphous fractions for Cellulose I structure, respectively. Iα mass fractions were calculated using absorbency data from FTIR spectrums recorded in the range 4,000–650 cm-1 as an average of eight scans at 4 cm−1 resolution (Perkin-Elmer,
Spectrum One). The following formula (2) described by Yamamoto et al. (1996) was used in calculations: f α ¼ 2:55 f ır 0:32
ð2Þ
where fα is the Iα mass fraction and fır is the absorbency ratio of 750 cm−1 to its sum with 710 cm−1, corresponding to absorbencies of Iα and Iβ phases. Freeze-dried bacterial cellulose samples were coated with platinum and viewed under field emission scanning electron microscope (JEOL, JSM 7000F) for investigation of morphological structure. The effect of cultivation method on thermal behavior of synthesized bacterial cellulose was studied by thermogravimetric analyzer (Perkin Elmer, Diamond TG/DTA). Sample size was within 5–10 mg. The weight loss was recorded under N2 atmosphere in 30–700 °C temperature range at heating rate of 10 °C/min.
Results Isolation and characterization of strains After spreading and repeated streaking of enriched waste samples on HS agar plates, 329 single isolates were selected according to distinctive colony morphologies. Selected isolates formed smooth, circular, convex, mucous, and cream- to beige-colored colonies on HS agar (Sievers and Swings 2005; Nguyen et al. 2010). The majority of the isolates originated from fruits, either in fresh or rotten state. One hundred thirtynine of these isolates were found to show similar characteristics with acetic acid bacteria in that their gram reactions were negative and they were rod shaped. The majority of the strains, i.e., 114 of 139, were non-motile. For 39 of 139 isolates, CaCO3 clear zones appeared on agar plates containing CaCO3–ethanol medium, which identified these isolates as members of Acetobacteraceae family. Four of the acidpositive isolates were classified as Gluconacetobacter sp. due to their deficiencies in acetic acid overoxidation test (Dellaglio et al. 2005; Sievers and Swings 2005). Of 35 remaining isolates, only nine strains, with given codes listed in Table 1, exhibited cellulose production. Production capacities were in the range of 0.19–4.70 g/l that were comparable to the productivities of reference strains, i.e., 3.73 g/l and 2.01 g/l for G. xylinus LMG 18788 and G. hansenii DSM 5602, respectively. Thus, out of 329 isolates, only nine isolates were subjected to extensive biochemical testing that provided the responses shown in Table 1. Mostly similar responses were recorded for reference and isolated strains in biochemical tests. Accordingly, all cellulose producer strains were oxidase negative and catalase positive. While all strains were tolerant to the presence of 0.35 % acetic
Appl Microbiol Biotechnol Table 1 Biochemical characteristics of cellulose producing isolates and reference strains Characteristics
AS6
AS7
AS14
AA4
AU9
TES2
DS1
P2A
ESU8
LMG 18788
DSM 5602
Catalase Oxidase Indole production Brown pigmentation H2S formation Growth in the presence of 0.35 % acetic acid 30 % D -Glucose Growth on methanol Urea utilization Sodium citrate utilization Acetate oxidation Lactate oxidation Gelatin liquefaction Ketogenesis of glycerol Nitrate reduction Growth on mannitol agar
+ − − − −
+ − − − −
+ − − − −
+ − − − −
+ − − − −
+ − − + −
+ − − − −
+ − − − −
+ − − − −
+ − − − −
+ − − − −
+ − − − − + − − − − +
+ − − − − + − − + − +
+ − − − − + − − + − +
+ − − − − + + − − − +
+ − − − + + + − − − +
+ − − − − + + − + − +
+ − − − − + + − − − +
+ − − − − + + − − − +
+ − − − − + + + + − +
+ − − − − + + − + − +
+ − − − wa + + − + − +
− + −
− + −
w + +
− + +
− + +
− + +
− + −
+ + −
+ + +
+ + −
− + −
+ + − − + − + −
+ + w − + − + −
+ + − − w − + −
+ + + − − − + −
+ + + − w − − −
+ + + − + − − −
+ + + + + − − +
+ + + + + + + +
+ + + − − − + −
+ + + − + − + +
+ + + − + + + −
Growth on glutamate agar Arginine dehydrolase Lysine decarboxylase Acid production from: Glucose Sucrose Fructose Lactose Galactose Maltose Mannose Xylose a
w denotes a weak positive response
acid, none of them could grow in the presence of 30 % D glucose. Negative reactions were observed for indole production, citrate and urea utilization, H2S formation, growth on methanol, and nitrate reduction. Strains could not grow at incubation temperatures below 20 °C and above 40 °C. Cell growth was not observed when medium pH was below 3 and above 8.0. Incubation temperature of 25–35 °C and pH of 4.0–7.0 were optimal ranges for maximization of cell growth. All these characteristics were in accordance with the previous reports on characterization of Acetobacter and Gluconacetobacter spp. (Yamada 2000; Lisdiyanti et al. 2001; Jojima et al. 2004; Kerters et al., 2006; Zahoor et al. 2006). All strains showed differential biochemical traits that aided in their identification in genus level. Strain TES2, isolated from grape vinegar, was the only strain that produced watersoluble brown pigment on CaCO3 agar plates. As this strain
provided positive results for acid production from D -fructose and grew well on mannitol agar, it was classified within Gluconacetobacter genus (Sievers and Swings, 2005). Liquefaction of gelatin was positive for strain ESU8, which was in contradiction to the previous literature reports concerning acetic acid bacteria (Asai and Shoda 1958; Gossele et al. 1984; Lisdiyanti et al. 2001; Sievers and Swings, 2005). The inexistence of a cellulose-producing strain with positive gelatinase activity led us to conclude that isolate ESU8 was either a G. xylinus or G. hansenii spp. Isolates coded AS6, AS7, and AS14 from vinegar fermentation waste were classified as Gluconacetobacter sp. according to their negative responses in lactate oxidation test (Yamada et al. 1976; Kojima et al. 1998). Gluconacetobacter sp. AS6 was classified as a G. xylinus strain due to its negative result in dihydroxyacetone production from glycerol. Gluconacetobacter sp. AS14 provided positive results in lysine decarboxylation and growth on
Appl Microbiol Biotechnol
glutamate agar similar to certain strains of G. xylinus as reported by Navarro and Komagata (1999). Isolates AA4 and AU9 from apple and grape vinegar showed almost exact biochemical characteristics with G. hansenii DSM 5602. Only isolates coded DS1 and P2A could produce acid from lactose. These strains exhibited similarity to G. xylinus LMG 18788 in acid production from xylose. Though biochemical testing provided some foresight into phenotypic characterization, identification at species level required the utilization of molecular methods such as sequence analysis of 16S rRNA gene (Yamada 2000). Prior to 16S rRNA sequencing, strains were tested for the sustainability of bacterial cellulose production over long-term preservation. Within a period of 1 year, only the freeze stocks of P2A could preserve their production capacities, while for the rest of isolates gradual decreases were observed. Thus, P2A was the only isolate involved in further experimentation. 16S rRNA sequence analysis of isolate P2A provided a nearly complete gene sequence (1,405 bp) which showed 99.8 % and 99.7 % similarities with sequences of G. hansenii NBRC 14820 and ATCC 23769. These percentages accounted for three and four base changes, respectively. The lowest similarity was recorded as 98.3 %, corresponding to 24 base changes with Gluconacetobacter intermediu s LMG 1689. Thus, the strain was named as G. hansenii P2A and deposited in the Centre for Research and Application of Culture Collections of Microorganisms (KUKENS), Turkey with the code KUEN 1606. 16S rRNA gene data was uploaded in GenBank database with the accession no. KF155166. The 16S rRNA gene sequence of G. hansenii P2A and similar sequences of Gluconacetobacter sp. retrieved from primary databanks (Gen Bank, EMBL, and DDBJ) were used in construction of the phylogenetic tree shown in Fig. 1. The neighbor-joining tree, which was constructed using Geneious Pro 5.6.6 software, placed P2A within G. hansenii cluster. Fig. 1 A phylogenetic tree based on 16S rRNA sequences constructed by the neighbor-joining method
Effect of cultivation conditions on bacterial cellulose production Under static cultivation conditions in HS medium, G. hansenii P2A produced 1.89±0.08 g/l cellulose. Over a period of 1 week, 80.7 % of glucose was consumed to produce bacterial cellulose with 0.12±0.31 g/g yield. In an attempt to increase cellulose production capacity of G. hansenii P2A, shaken-flask experiments were conducted at 120, 150, and 200 rpm. Accordingly, shaking at a rate of 120 rpm led to an increment of 1.17-fold in bacterial cellulose production. The produced amount of cellulose was stable during five consecutive batches at an average value of 2.21±0.03 g/l corresponding to an average yield of 0.12±0.20 g/g. The stability of cellulose production implied that mutation was not effective at this shaking rate. However, at shaking rates of 150 and 200 rpm, adverse effects were observed in productivity and yield. Even in the first batches, the amount of synthesized cellulose was reduced significantly, i.e., to 0.52±0.04 and 0.27±0.09 g/l, for 120 and 150 rpm, respectively. Cellulose productivity decreased below 0.1 g/l by the end of the third cycle for both shaking rates. The decline in cellulose production was ascribed to the appearance of cellulose non-producing mutants, a phenomenon described as early as 1954 by Schramm and Hestrin. Colony counts revealed the high frequency of cellulose-less mutants at rates of 150 and 200 rpm. Figure 2 shows the morphological differences between colonies of G. hansenii P2A and its cellulose-less mutants. Mutant colonies were discriminated by much bigger, round, and mucous colonies on HS agar plates. The morphology of mutant colonies was in accordance with the descriptions provided elsewhere (Schramm and Hestrin 1954; Krystynowicz et al. 2002; Nguyen et al. 2010). Mutant and wild-type colonies were counted separately and mutant ratios were calculated for each batch at a corresponding shaking rate. Results are summarized in Table 2.
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Morphological and structural characterization of bacterial cellulose
Fig. 2 Colony morphology of native Gluconacetobacter hansenii P2A strains and its cellulose non-producing mutants. a Wild type, b celluloseless mutants
Accordingly, cell growth increased proportionally with shaking rate. At 120 rpm, only 2 % of the viable cells were transformed to cellulose-less forms by the end of the fifth cycle, confirming the shear resistance. The increased cell density and the low mutation rate justified for the enhancement in cellulose production. However, at shaking rates of 150 and 200 rpm, mutant colonies appeared just by the first cycle. Since previous batch was used as inoculum for the next batch, mutant ratio increased gradually during consecutive cycles, reaching as high as 64 %, by the end of the fifth cycle at 200 rpm. Mutant colonies were incubated statically in HS medium to check if mutation was reversible. Unfortunately, pellicle formation was never observed, and thus cellulose production ability was not retrievable. Agitated culture conditions were realized at 120 rpm as it minimized the rate of mutant formation. Under the oxygen supply of 0.5 vvm, volumetric mass transfer coefficient (kLa) was calculated as 23 h−1 (data not shown). These conditions were sufficient to attain 30 % of oxygen saturation within the culture medium. By the end of 72 h, the amount of bacterial cellulose synthesized by G. hansenii P2A reached to its highest value, i.e., 3.25±0.11 g/l that corresponded to 0.17± 0.01 yield. Mutant ratio was recorded as 4.0 %, notably higher than shaken culture. The increase in mutant formation was attributed to the combined effect of shear exerted on bacterial cells by the turbine impeller and efficient aeration that led to increased free cell (i.e., not embedded in cellulose) population (Krystynowicz et al. 2002; Park et al. 2003a).
Cultivation conditions affected morphological and structural properties of bacterial cellulose synthesized by G. hansenii P2A. Bacterial cells built a thick biofilm in static culture, while non-uniform cellulose globules were synthesized in agitated and shaken cultures. Morphological structure was studied in detail using SEM images provided in Fig. 3. Accordingly, the cellulosic mat produced under static conditions exhibited an ordered and dense network of fibrils with diameters as thin as 8–10 nm. The cross-sectional view (Fig. 3a) showed that the mat was composed of interconnected layers. On the other hand, synthesized cellulose accumulated in the form of a much looser clump of disordered short and thin fibrils under agitated cultivation conditions (Fig. 3c, d). The difference in aggregation behavior of cellulose fibrils induced by cultivation conditions led to changes in crystallinity as shown by the X-ray diffractograms (Fig. 4a–c). All diffraction profiles showed peaks around 2θ =14.70°, 16.6°, and 22.50° which correspond to typical peaks of 101, 101 , and 002 planes of Cellulose I crystal structure (Ford et al. 2010). Relative crystallinity index of cellulose samples were calculated as 78.7, 72.6, and 77.3 % by Segal method (Eq. 1) for static, shaken, and agitated conditions, respectively. The changes in relative peak intensities of 101 and 101 planes indicated that crystal phase ratios were also different. Mass fractions of crystal phases were calculated using FTIR spectrums that are shown in Fig. 5. The absorbencies of spectral bands at 750 and 710 cm−1 representing Iα and Iβ phases, respectively, were used in calculation of the mass fractions of Iα phase of cellulose samples. FTIR spectrums were also used for description functional groups. The spectral bands of all three bacterial cellulose samples were almost identical and exhibited characteristic bands of Cellulose I structure as reported elsewhere (Bertocchi et al. 1997; Rani et al. 2011; Gea et al. 2011). However, the intensity of spectral band at 895 cm−1 corresponding to β glycosidic linkages (Wong et al. 2009) was suppressed for samples synthesized under agitated conditions. The declines in the proportion of β(1–4) linkages implied that synthesized celluloses had
Table 2 The effect of shaking rate on the conversion of Gluconacetobacter hansenii P2A into non-cellulose-producing mutants Shaking rate
1st Batch
2nd Batch
3rd Batch
4th Batch
(rpm)
cfu/ml
m.r.
cfu/ml
m.r.
cfu/ml
m.r.
cfu/ml
m.r.
cfu/ml
m.r.
0 120 150 200
2.3 × 108 6.2 × 108 1.4 × 109 1.1 × 1010
0 0 0.05 0.10
2.8 × 108 9 × 108 1 × 1010 1.5 × 1010
0 0 0.06 0.15
3.1 × 108 1.9 × 109 1.6 × 1010 2.2 × 1011
0 0.01 0.09 0.24
6.4 × 108 5 × 109 2.3 × 1010 4.7 × 1011
0 0.02 0.10 0.43
7 × 108 1.09 × 1010 3.1 × 1010 6.8 × 1011
0 0.02 0.19 0.64
m.r. mutant ratio calculated as the number of mutant cells divided by the total number of cells
5th Batch
Appl Microbiol Biotechnol Fig. 3 SEM images showing BC produced under stationary culture (a, b) and shaken culture (c, d)
lower molecular weights with respect to the sample synthesized under stationary conditions. The intensity ratios of 750 cm−1 and 710 cm−1 bands were also reduced in agitated synthesis. Through the use of Eq. 2, Iα mass fractions were calculated as 0.88, 0.81, and 0.79 for static, shaken, and agitated cultivation conditions, respectively. Thus, enhanced agitation led to the increase of mass fraction of thermally more stable Iβ crystal phase (Yamamoto et al. 1996). The increase in the mass fraction of Iβ phase suggested a proportional enhancement in thermal stability, which was investigated by thermogravimetric analysis. The thermal behavior of bacterial celluloses synthesized by G. hansenii P2A under different cultivation conditions are shown in Fig. 6. TGA curves of samples produced under static, shaken, and agitated conditions showed single step degradations with maximum decomposition temperatures of 313.3, 320.6, and 332.7 °C, respectively. The thermal stability increased when agitation was applied in culture medium, which confirmed the
gradual increase in Iβ phase content. The residual matter was about 20 % for cellulose produced under static conditions, while 29 % residue was obtained for cellulose produced under shaken conditions. The increase in char ratio was attributed to impurities that also provided a small band in corresponding FTIR spectrum (i.e., at 1,700 cm−1 in Fig. 5c). In the case of agitated culture, the thermal degradation was yet incomplete at 680 °C, which might have aroused from the large sample size with respect to other cellulose samples (6.5 mg vs. 10 mg) or from the substantial increase in decomposition resistance.
Discussion With this study, nine cellulose-producing strains, with production capacities between 0.19 and 4.70 g/l, were isolated from waste and fresh vinegar samples and fruits. Although the recent classification of acetic acid bacteria groups cellulose-
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Fig. 5 FTIR spectra of bacterial cellulose samples synthesized in a static culture, b agitated culture, and c shaken culture at 120 rpm by Gluconacetobacter hansenii P2A
Fig. 4 X-ray diffraction spectra of bacterial celluloses synthesized under different cultivation conditions by Gluconacetobacter hansenii P2A: a static culture; b shaken culture; c agitated culture at 120 rpm
secreting strains under Gluconacetobacter genus (Kersters et al. 2006), the existence of several other bacterial species having this phenotypic trait (Czaja et al. 2006) convinced us to carry out morphological and biochemical inspection. Results proved that all isolates complied with Gluconacetobacter genus of Acetobacteraceae family. Even though biochemical testing was not sufficient for identification at species level, interesting and definitive results were obtained for some of the isolates. The most significant result was the liquefaction of gelatin by Gluconacetobacter sp. ESU8. To our knowledge, this was the first report indicating a Gluconacetobacter sp. with positive gelatinase activity. The production capacity of ESU8, i.e., 1.38±0.01 g/l, was similar to the previously reported values for G. hansenii (Park 2003b) and G. xylinus strains (Toyosaki et al. 1995), which implied that this strain could be a corresponding subspecies. However, like all
isolates other than P2A, the cellulose productivity of ESU8 diminished during short-term storage and cell recovery was very poor after resuscitation of freeze stocks. Thus, further research involving 16S rRNA sequencing could not be conducted for these isolates. Gullo et al. (2012) have defined strong relations between subculture frequency and persistence of phenotypic traits, which led us to discuss that 2 weeks of subculturing time was inappropriate for these isolates. Use of cryoprotectants other than glycerol, such as mannitol, malt extract, or sorbitol, might construct another strategy for preservation of viability of freezed cells (Ndoye et al. 2009). These strategies will be implemented in further studies involving isolation of acetic acid bacteria. As a result, isolate P2A was selected as the species with the highest potential for commercial applications owing to its high phenotypic stability during short-term preservation and effective cell recovery after long-term storage, in addition to its sustainable production capacity (Stanbury et al. 1995) of 1.89±0.08 g/l under static cultivation conditions. This value was quite similar to the 2.01 g/l cellulose production by G. hansenii DSM 5602, which was used as reference strain in biochemical testing. The nearly complete 16S rRNA sequence of P2A identified this strain as G. hansenii P2A and thus confirmed the resemblance. Though not a hyperproducer, the production capacity of G. hansenii P2A was found to be significantly higher than the production capacity of G. xylinus K3 (0.217±0.027 g/l) reported by Nguyen et al. (2010) and G. hansenii PJK (0.35 g/l) reported by Park et al. (2003b). Yet, it is possible to enhance production capacity by manipulation of medium composition (Oikawa et al. 1995; Son et al. 2003) or cultivation conditions (Park et al. 2003a; Czaja et al. 2004). Our investigations on cultivation at shaken culture conditions proved that 120 rpm maximized cell growth and cellulose production, while keeping mutation rate at minimum over five consecutive batches. Literature reports on investigation of the effects of shaking rate on bacterial cellulose production by Gluconacetobacter sp. confirm the existence of an optimum shaking rate. Son et al. (2002) have reported that 150 rpm maximized cellulose production by Acetobacter xylinum KJ-1
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Fig. 6 TGA curves of bacterial cellulose samples synthesized in static culture (solid line), agitated culture (dotted line), and shaken culture (dashed line) at 120 rpm by Gluconacetobacter hansenii P2A
similarly to Mohite et al. (2012), who have determined that shaking at 200 rpm advances cellulose production by G. hansenii NCIM 2529 strain by 28 %. Though the detected optimum shaking rate in this study was relatively slower, the productivity of G. hansenii P2A was yet very high with respect to other G. hansenii strains reported in literature (Park et al. 2003b), i.e., 2.21±0.03 g/l versus 0.6 g/l, for the same initial medium composition. In contrast to previous literature (Shoda and Sugano 2005), agitation and efficient aeration upgraded the productivity of G. hansenii P2A to 3.25±0.11 g/l, which also emphasized the importance of efficient pH control for this strain. The utilization of supplementary materials such as ethanol and agar were not necessary for the maintenance cellulose producing ability as proposed by Park et al. (2003a) and Bae et al. (2004) indicating the shear resistance of the isolated strain. Culture conditions caused morphological and structural changes in synthesized cellulose by G. hansenii P2A. While significant reductions were observed in fibril dimensions, the crystallinity index of synthesized bacterial cellulose was only slightly affected by shaking and agitation at 120 rpm. The relative crystallinity index of bacterial cellulose can reach as high as 90 %, when bacteria are cultivated under static conditions (Chen et al. 2010; Siró and Plackett 2010), and for a given strain, 5–15 % decline is acceptable for agitated cultivation conditions (Watanabe et al. 1998; Czaja et al. 2004). Bacterial cellulose synthesized by G. hansenii P2A under 120 rpm
agitation exhibited only 1.5 % decline in relative crystallinity index. This was attributed to both shear resistance and attachment of some portion of cells on the agitator and probes, a phenomenon employed in disc bioreactors (Krystynowicz et al. 2002). Shaking and agitation led to 8 % and 10 % increments in Iβ phase ratio, respectively. The increase was accompanied by enhanced thermal resistance with a shift of almost 20 °C in maximum decomposition temperature while the nature of the curve was still typical of pure bacterial cellulose decomposing in a single degradation step that starts usually about 280– 300 °C (George et al. 2005; Gea et al. 2011; Wong et al. 2009). All results proved that G. hansenii P2A could be a candidate microorganism for commercial production of bacterial cellulose. Even though the yield was slightly lower than the 20 % limit (Raspor and Goranovic 2008), further studies are planned for the development of an optimized medium and determination of optimal culture conditions such as pH and temperature aiming for enhancement of the yield and production capacity.
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Appl Microbiol Biotechnol Segal L, Creely JJ, Martin AE Jr, Conrad CM (1959) An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Text Res J 29:786–794 Shah J, Brown RM Jr (2005) Towards electronic paper displays made from microbial cellulose. Appl Microbiol Biotechnol 66:352– 355 Shoda M, Sugano Y (2005) Recent advances in bacterial cellulose production. Biotechnol Bioprocess Eng 10:1–8 Sievers M, Swings J (2005) Family II: Acetobacteraceae. In: Brenner DJ, Krieg NR, Staley JT (eds) Bergey’s manual of systematic bacteriology, vol 2, 2nd edn. Springer, New York, pp 41–96 Siró I, Plackett D (2010) Microfibrillated cellulose and new nanocomposite materials: a review. Cellulose 17:459–494 Son C, Chung S, Lee J, Kim S (2002) Isolation and cultivation characteristics of Acetobacter xylinum KJ-1 producing bacterial cellulose in shaking cultures. J Microbiol Biotechnol 12:722–728 Son HJ, Kim HG, Kim KK, Kim HS, Kim YG, Lee SJ (2003) Increased production of bacterial cellulose by Acetobacter sp. V6 in synthetic media under shaking culture conditions. Bioresour Technol 86:215– 219 Stanbury PF, Whitaker A, Hall SJ (1995) Principles of fermentation technology, 2nd edn. Butterworth Heinemann, Oxford Toyosaki H, Kojima Y, Tsuchida T, Hoshino K, Yamada Y, Yoshinaga F (1995) The characterization of an acetic acid bacterium useful for producing bacterial cellulose in agitation cultures: the proposal of
Acetobacter xylinum subsp. sucrofermentans subsp. nov. J Gen Appl Microbiol 41:307–314 Vandamme EJ, De Baets S, Vanbaelen A, Joris K, De Wulf P (1998) Improved production of bacterial cellulose and its application potential. Polym Degrad Stab 59:93–99 Watanabe K, Tabuchi M, Ishikawa A, Morinaga Y, Yoshinaga F (1998) Structural features and properties of bacterial cellulose produced in agitated culture. Cellulose 5:187–200 Wong SS, Kasapis S, Tan YM (2009) Bacterial and plant cellulose modification using ultrasound irradiation. Carbohydr Polym 77: 280–287 Yamamoto H, Horii F, Hirai A (1996) In situ crystallization of bacterial cellulose. II. Influences of polymeric additives with different molecular weights on the formation of celluloses Iα and Iβ at the early stage of incubation. Cellulose 3:229–242 Yamada Y, Okada Y, Kondo K (1976) Isolation and characterization of ‘polarly flagellated intermediate strains’ in acetic acid bacteria. J Gen Appl Microbiol 22:237–245 Yamada Y (2000) Transfer of Acetobacter oboediens and Acetobacter intermedius to the genus Gluconacetobacter as Gluconacetobacter oboediens comb. nov. and Gluconacetobacter intermedius comb. nov. Int J Syst Evol Microbiol 50:2225–2227 Zahoor T, Siddique F, Farooq U (2006) Isolation and characterization of vinegar culture (Acetobacter aceti) from indigenous sources. Brit Food J 108:429–439
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Isolation and characterization of an efficient bacterial cellulose producer strain in agitated culture: Gluconacetobacter hansenii P2A Yasar Andelib Aydın & Nuran Deveci Aksoy
Received: 8 July 2013 / Revised: 10 September 2013 / Accepted: 26 September 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract In this study, typical niches of acetic acid bacteria were screened for isolation of cellulose producer strains. Hestrin Schramm broth was used as enrichment and production media. Only nine out of 329 isolates formed thick biofilms on liquid surface and were identified as potential cellulose producers. Physiological and biochemical tests proved that all cellulose producers belonged to Gluconacetobacter genus. Most productive and mutation-resistant strain was subjected to 16S rRNA sequence analysis and identified as Gluconacetobacter hansenii P2A due to 99.8 % sequence similarity. X-ray diffraction analysis proved that the biofilm conformed to Cellulose I crystal structure, rich in Iα mass fraction. Static cultivation of G. hansenii P2A in HS medium resulted with 1.89±0.08 g/l of bacterial cellulose production corresponding to 12.0±0.3 % yield in terms of substrate consumption. Shaking and agitation at 120 rpm aided in enhancement of the amount and yield of produced cellulose. Productivity and yield reached up to 3.25± 0.11 g/l and 17.20±0.14 % in agitated culture while a slight decrease from 78.7 % to 77.3 % was observed in the crystallinity index. Keywords Bacterial cellulose . Gluconacetobacterhansenii . 16S rRNA sequence analysis . Mutation
Introduction Bacterial cellulose is the network of cellulose fibrils secreted by certain strains of bacteria, belonging mostly to the Gluconacetobacter genus of Acetobacteraceae family (Vandamme et al. 1998). The members of this family are Y. A. Aydın (*) : N. D. Aksoy Chemical Engineering Department, Istanbul Technical University, 34469, Sarıyer Istanbul, Turkey e-mail: [email protected]
obligate aerobes that have the ability to convert ethanol to acetic acid and, as a consequence, can grow at low pH levels (Kersters et al. 2006; Hwan et al. 2004). Gluconacetobacter sp. can be isolated from a variety of sugary, acidic, and alcoholic environments such as fruits, flowers, vinegar, Kombucha, beer, tequila, and wine (Gossele et al. 1984; Toyosaki et al. 1995; Lisdiyanti et al. 2001; Dellaglio et al. 2005; Nguyen et al. 2010). It is accepted that the cellulosic biofilm produced by these bacteria functions not only by positioning the cells at the surface, where oxygen tension is high, but also by protecting them from the harmful effects of ultraviolet and enemies, yet with a continuous supply of nutrients through diffusion (Iguchi et al. 2000). More recently, Kanchanarach et al. (2010) related polysaccharide formation with acetic acid resistance and proved that the extracellular biofilm produced by rough colonies of Acetobacter pasteurianus strains (IFO3283, SKU1108, MSU10) served for the hindrance of acetic acid diffusion to the cytoplasmic membrane. In addition to its biological function in the survival of microorganisms, bacterial cellulose offers a vast variety of end uses due to its unique material properties. While plant cellulose contains impurities such as lignin, pectin, hemicellulose, and other metabolic products, bacterial cellulose is completely pure and highly crystalline (Raspor and Goranovic 2008). Bacterial cellulose is also renown with its very high water absorptivity reaching up to 200 times its weight (Krystynowicz et al. 2002) and superior mechanical stability with respect to plant-derived cellulose fibers (Shoda and Sugano 2005). The fiber dimensions are also significantly lower for bacterial cellulose, practically in the order of a few nanometers. These special properties have led to the use of this material in a diversity of applications especially in medicine, health, and materials sciences. Several applications were suggested commercially and scientifically of which artificial blood vessels (Klemm et al. 2011), skin and tissue
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replacements, acoustic diaphragms (Czaja et al. 2006), electronic displays (Shah and Brown, Jr. 2005), and reinforcement agent in paper and nanocomposites (Siró and Plackett 2010; Chen et al. 2010) are a few. Despite these high-tech end uses, bacterial cellulose production is yet limited by a series of factors affecting process economics (Moon et al. 2006). Conventional method of bacterial cellulose production relies on the static cultivation of bacteria, which is an inefficient, time-consuming, and labor-intensive production scheme (Bae and Shoda 2005). The production capacity of static cultivation is usually very low due to space requirements and insufficient supply of oxygen (Kouda et al. 1997). However, this scheme might be advantageous when pellicle-formed cellulose is required as such in skin replacements or a relatively higher degree of crystallinity is preferable (Raspor and Goranovic 2008). Although enhanced oxygen transfer occurring in agitated fermentation should theoretically increase the rate of cellulose production, most often reduced yields were reported (Shoda and Sugano 2005). The shear stress exerted on the bacterial cells causes spontaneous mutation of Gluconacetobacters by deactivating essential enzymes involved in cellulose synthesis, i.e., phosphoglucomutase and uridine diphosphoglucose pyrophosphorylase (Kornmann et al. 2003; Nguyen et al. 2010). Mutant strains may also show an enhanced ability for accumulation of water-soluble polysaccharides such as acetan (Ross et al. 1991), levan (Bae and Shoda 2005), and xylan (Moon et al. 2006) that use the same starter molecule with bacterial cellulose, i.e., uridine diphosphate glucose, leading also to reduced cellulose production rates. Strategies applied for inhibition of mutation or lowering its effects include addition of supplements such as ethanol (Park et al. 2003a) and agar (Bae et al. 2004), selection of highly productive mutation-resistant strains (Toyosaki et al. 1995), generation of non-acetan (Ishida et al. 2002) or non-ketogluconate (Vandamme et al. 1998) producing mutants, and development of cultivation strategies lowering shear, such as using different impeller types (Kouda et al. 1997), air lift reactors, and rotating disc type reactors (Krystynowicz et al. 2002; Shoda and Sugano 2005). In the current study, we aimed to isolate bacteria possessing high and sustainable ability to produce cellulose from typical niches of acetic acid bacteria. Isolates were characterized in genus level by biochemical and morphological tests. The strain that exhibited highest stability during long-term preservation was subjected to 16S rRNA sequence analysis and characterized in species level. Cellulose production was tested under static, shaken, and agitated conditions. Morphological and structural changes in the synthesized bacterial cellulose were investigated by X-ray diffraction, Fourier transform infrared (FTIR), scanning electron microscopy (SEM), and thermogravimetric analysis (TGA).
Materials and methods Isolation and characterization of microorganisms All chemicals and reagents were either of analytical or biochemical grade. While vinegar fermentation waste was kindly provided by Kemal Kukrer A.S., Turkey, vinegar samples, and fresh and rotten fruits (apples, grapes, apricots, pineapples, chestnuts, and plums) were collected from local markets. Samples were incubated at 28 °C in Hestrin–Schramm (HS) medium (2.0 % D -glucose, 0.5 % peptone, 0.5 % yeast extract, 0.27 % Na2HPO4, and 0.115 % citric acid) for 7 days (Schramm and Hestrin 1954). HS medium was supplemented with 50 mg/l cycloheximide for prevention of the growth of undesired microorganisms such as yeasts. Enriched samples of 0.1 ml volume were streaked onto HS agar (1.5 % agar) plates. Growth was monitored during 3 days of incubation at 28 °C. With repeated streaking, white- to cream-colored mucous colonies were isolated. Isolates were verified as acetic acid bacteria with visualization of CaCO3 clear zones in plates containing CaCO3–ethanol medium (0.05 % D -glucose, 0.3 % peptone, 0.5 % yeast extract, 1.5 % CaCO3, 1.2 % agar, and 1.5 % ethanol). Gluconacetobacter sp. and Acetobacter sp. were further selected by a positive result in overoxidation of acetic acid test as proposed by Dellaglio et al. (2005). Formation of a biofilm at the air vicinity of culture broth after a week of static incubation at 30 °C was accepted as an indication of cellulose production. The pellicle, either stagnant or fragile, was removed by centrifugation, washed with distilled water, retained in 0.1 M NaOH for 20 min at 80 °C, and finally neutralized in 0.1 M acetic acid. After extensive washing with distilled water, any remaining material was either vacuum dried at 0.1 bar and 40 °C or freeze dried at −50 °C and 0.04 mbar. Resistance of the biofilm to this treatment proved conformity to cellulose structure. Strains with cellulose production ability were subjected to further biochemical testing according to the methods described by Asai (1958), Yamada et al. (1976), and Lidiyanti et al. (2001). Gluconacetobacter xylinus LMG 18788 and Gluconacetobacter hansenii DSM 5602 were used as reference strains for biochemical testing. All isolates and reference strains were preserved by freezing in glycerol at −20 °C and monthly checked for strain stability. Sequencing of 16S rRNA gene Most productive strain was identified by 16S rRNA sequencing. Accordingly, the 16S rRNA gene was amplified by PCR with 20F and 1492R primers. The purified PCR products were sequenced directly by 27F, 357F, 530F, 343R, 907R, 1100R, 1392R, and 1492R universal primers using Beckman Coulter sequence analysis tool (CEQ™ DTCS Quick Start Kit, Beckman Coulter). A BLAST search of the determined gene sequence provided highly similar sequences from the same
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phylogenetic group, which were aligned in CLUSTAL Omega (Goujon et al. 2010). A phylogenetic tree was constructed by neighbor-joining method using Geneious 5.6.6 software. The 16S rRNA gene sequence was deposited in GenBank database with the accession number KF155166. The isolate was lyophilized and deposited in Centre for Research and Application of Culture Collections of Microorganisms (KUKENS), Turkey with the reference number KUEN 1606. Culture conditions Stock cultures (1 ml) were grown in 100 ml HS medium at 30 °C under static conditions for 48 h. Vigorous shaking was applied for detachment of bacterial cells and supernatant of this broth was used as seed for batch cellulose production experiments. Inoculation ratio, pH, and cultivation temperature were 10 %, 5.0, and 28 °C, respectively. Static and shaken flask experiments were conducted in 250-ml flasks with 50 ml culture broth, while a 1.5-l jar fermenter (Electrolab, FerMac 200, 1 l working volume) equipped with pH, temperature, and DO (dissolved oxygen) control was used for agitated culture conditions. The pH adjustment was realized by automatic addition of 1 N NaOH and 1 N HCl. The filtered oxygen was supplied at 0.5 vvm. Cultivation periods were a week, 5 days, and 3 days for static, shaken, and agitated conditions, respectively. Samples were taken at definite time intervals to analyze cell growth, cellulose production, and glucose consumption. DNS method was used for glucose assays (Miller 1959). Cell growth was monitored on HS agar plates where any cellulose negative mutants were easily detected and counted by morphological differences. Mutation resistance was tested in five consecutive batches at shaking rates of 120, 150, and 200 rpm in which previous batches were used as inoculants. All experiments were performed in triplicates to ensure the accuracy of the results. Analysis of properties of bacterial cellulose Crystal structure of bacterial cellulose samples were analyzed between 2θ =5–40° at 0.002° angular intervals using an X-ray diffractometer (PANalytical, XPERT PRO). Relative crystallinity index was calculated according to the formula constructed by Segal (1959): CrI ¼
I002 −Iam I002
100
ð1Þ
where CrI is the relative crystallinity index, I002 is the diffraction intensity of 002 plane, and Iam is the intensity of the peak at 2θ =18°, corresponding to crystal and amorphous fractions for Cellulose I structure, respectively. Iα mass fractions were calculated using absorbency data from FTIR spectrums recorded in the range 4,000–650 cm-1 as an average of eight scans at 4 cm−1 resolution (Perkin-Elmer,
Spectrum One). The following formula (2) described by Yamamoto et al. (1996) was used in calculations: f α ¼ 2:55 f ır 0:32
ð2Þ
where fα is the Iα mass fraction and fır is the absorbency ratio of 750 cm−1 to its sum with 710 cm−1, corresponding to absorbencies of Iα and Iβ phases. Freeze-dried bacterial cellulose samples were coated with platinum and viewed under field emission scanning electron microscope (JEOL, JSM 7000F) for investigation of morphological structure. The effect of cultivation method on thermal behavior of synthesized bacterial cellulose was studied by thermogravimetric analyzer (Perkin Elmer, Diamond TG/DTA). Sample size was within 5–10 mg. The weight loss was recorded under N2 atmosphere in 30–700 °C temperature range at heating rate of 10 °C/min.
Results Isolation and characterization of strains After spreading and repeated streaking of enriched waste samples on HS agar plates, 329 single isolates were selected according to distinctive colony morphologies. Selected isolates formed smooth, circular, convex, mucous, and cream- to beige-colored colonies on HS agar (Sievers and Swings 2005; Nguyen et al. 2010). The majority of the isolates originated from fruits, either in fresh or rotten state. One hundred thirtynine of these isolates were found to show similar characteristics with acetic acid bacteria in that their gram reactions were negative and they were rod shaped. The majority of the strains, i.e., 114 of 139, were non-motile. For 39 of 139 isolates, CaCO3 clear zones appeared on agar plates containing CaCO3–ethanol medium, which identified these isolates as members of Acetobacteraceae family. Four of the acidpositive isolates were classified as Gluconacetobacter sp. due to their deficiencies in acetic acid overoxidation test (Dellaglio et al. 2005; Sievers and Swings 2005). Of 35 remaining isolates, only nine strains, with given codes listed in Table 1, exhibited cellulose production. Production capacities were in the range of 0.19–4.70 g/l that were comparable to the productivities of reference strains, i.e., 3.73 g/l and 2.01 g/l for G. xylinus LMG 18788 and G. hansenii DSM 5602, respectively. Thus, out of 329 isolates, only nine isolates were subjected to extensive biochemical testing that provided the responses shown in Table 1. Mostly similar responses were recorded for reference and isolated strains in biochemical tests. Accordingly, all cellulose producer strains were oxidase negative and catalase positive. While all strains were tolerant to the presence of 0.35 % acetic
Appl Microbiol Biotechnol Table 1 Biochemical characteristics of cellulose producing isolates and reference strains Characteristics
AS6
AS7
AS14
AA4
AU9
TES2
DS1
P2A
ESU8
LMG 18788
DSM 5602
Catalase Oxidase Indole production Brown pigmentation H2S formation Growth in the presence of 0.35 % acetic acid 30 % D -Glucose Growth on methanol Urea utilization Sodium citrate utilization Acetate oxidation Lactate oxidation Gelatin liquefaction Ketogenesis of glycerol Nitrate reduction Growth on mannitol agar
+ − − − −
+ − − − −
+ − − − −
+ − − − −
+ − − − −
+ − − + −
+ − − − −
+ − − − −
+ − − − −
+ − − − −
+ − − − −
+ − − − − + − − − − +
+ − − − − + − − + − +
+ − − − − + − − + − +
+ − − − − + + − − − +
+ − − − + + + − − − +
+ − − − − + + − + − +
+ − − − − + + − − − +
+ − − − − + + − − − +
+ − − − − + + + + − +
+ − − − − + + − + − +
+ − − − wa + + − + − +
− + −
− + −
w + +
− + +
− + +
− + +
− + −
+ + −
+ + +
+ + −
− + −
+ + − − + − + −
+ + w − + − + −
+ + − − w − + −
+ + + − − − + −
+ + + − w − − −
+ + + − + − − −
+ + + + + − − +
+ + + + + + + +
+ + + − − − + −
+ + + − + − + +
+ + + − + + + −
Growth on glutamate agar Arginine dehydrolase Lysine decarboxylase Acid production from: Glucose Sucrose Fructose Lactose Galactose Maltose Mannose Xylose a
w denotes a weak positive response
acid, none of them could grow in the presence of 30 % D glucose. Negative reactions were observed for indole production, citrate and urea utilization, H2S formation, growth on methanol, and nitrate reduction. Strains could not grow at incubation temperatures below 20 °C and above 40 °C. Cell growth was not observed when medium pH was below 3 and above 8.0. Incubation temperature of 25–35 °C and pH of 4.0–7.0 were optimal ranges for maximization of cell growth. All these characteristics were in accordance with the previous reports on characterization of Acetobacter and Gluconacetobacter spp. (Yamada 2000; Lisdiyanti et al. 2001; Jojima et al. 2004; Kerters et al., 2006; Zahoor et al. 2006). All strains showed differential biochemical traits that aided in their identification in genus level. Strain TES2, isolated from grape vinegar, was the only strain that produced watersoluble brown pigment on CaCO3 agar plates. As this strain
provided positive results for acid production from D -fructose and grew well on mannitol agar, it was classified within Gluconacetobacter genus (Sievers and Swings, 2005). Liquefaction of gelatin was positive for strain ESU8, which was in contradiction to the previous literature reports concerning acetic acid bacteria (Asai and Shoda 1958; Gossele et al. 1984; Lisdiyanti et al. 2001; Sievers and Swings, 2005). The inexistence of a cellulose-producing strain with positive gelatinase activity led us to conclude that isolate ESU8 was either a G. xylinus or G. hansenii spp. Isolates coded AS6, AS7, and AS14 from vinegar fermentation waste were classified as Gluconacetobacter sp. according to their negative responses in lactate oxidation test (Yamada et al. 1976; Kojima et al. 1998). Gluconacetobacter sp. AS6 was classified as a G. xylinus strain due to its negative result in dihydroxyacetone production from glycerol. Gluconacetobacter sp. AS14 provided positive results in lysine decarboxylation and growth on
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glutamate agar similar to certain strains of G. xylinus as reported by Navarro and Komagata (1999). Isolates AA4 and AU9 from apple and grape vinegar showed almost exact biochemical characteristics with G. hansenii DSM 5602. Only isolates coded DS1 and P2A could produce acid from lactose. These strains exhibited similarity to G. xylinus LMG 18788 in acid production from xylose. Though biochemical testing provided some foresight into phenotypic characterization, identification at species level required the utilization of molecular methods such as sequence analysis of 16S rRNA gene (Yamada 2000). Prior to 16S rRNA sequencing, strains were tested for the sustainability of bacterial cellulose production over long-term preservation. Within a period of 1 year, only the freeze stocks of P2A could preserve their production capacities, while for the rest of isolates gradual decreases were observed. Thus, P2A was the only isolate involved in further experimentation. 16S rRNA sequence analysis of isolate P2A provided a nearly complete gene sequence (1,405 bp) which showed 99.8 % and 99.7 % similarities with sequences of G. hansenii NBRC 14820 and ATCC 23769. These percentages accounted for three and four base changes, respectively. The lowest similarity was recorded as 98.3 %, corresponding to 24 base changes with Gluconacetobacter intermediu s LMG 1689. Thus, the strain was named as G. hansenii P2A and deposited in the Centre for Research and Application of Culture Collections of Microorganisms (KUKENS), Turkey with the code KUEN 1606. 16S rRNA gene data was uploaded in GenBank database with the accession no. KF155166. The 16S rRNA gene sequence of G. hansenii P2A and similar sequences of Gluconacetobacter sp. retrieved from primary databanks (Gen Bank, EMBL, and DDBJ) were used in construction of the phylogenetic tree shown in Fig. 1. The neighbor-joining tree, which was constructed using Geneious Pro 5.6.6 software, placed P2A within G. hansenii cluster. Fig. 1 A phylogenetic tree based on 16S rRNA sequences constructed by the neighbor-joining method
Effect of cultivation conditions on bacterial cellulose production Under static cultivation conditions in HS medium, G. hansenii P2A produced 1.89±0.08 g/l cellulose. Over a period of 1 week, 80.7 % of glucose was consumed to produce bacterial cellulose with 0.12±0.31 g/g yield. In an attempt to increase cellulose production capacity of G. hansenii P2A, shaken-flask experiments were conducted at 120, 150, and 200 rpm. Accordingly, shaking at a rate of 120 rpm led to an increment of 1.17-fold in bacterial cellulose production. The produced amount of cellulose was stable during five consecutive batches at an average value of 2.21±0.03 g/l corresponding to an average yield of 0.12±0.20 g/g. The stability of cellulose production implied that mutation was not effective at this shaking rate. However, at shaking rates of 150 and 200 rpm, adverse effects were observed in productivity and yield. Even in the first batches, the amount of synthesized cellulose was reduced significantly, i.e., to 0.52±0.04 and 0.27±0.09 g/l, for 120 and 150 rpm, respectively. Cellulose productivity decreased below 0.1 g/l by the end of the third cycle for both shaking rates. The decline in cellulose production was ascribed to the appearance of cellulose non-producing mutants, a phenomenon described as early as 1954 by Schramm and Hestrin. Colony counts revealed the high frequency of cellulose-less mutants at rates of 150 and 200 rpm. Figure 2 shows the morphological differences between colonies of G. hansenii P2A and its cellulose-less mutants. Mutant colonies were discriminated by much bigger, round, and mucous colonies on HS agar plates. The morphology of mutant colonies was in accordance with the descriptions provided elsewhere (Schramm and Hestrin 1954; Krystynowicz et al. 2002; Nguyen et al. 2010). Mutant and wild-type colonies were counted separately and mutant ratios were calculated for each batch at a corresponding shaking rate. Results are summarized in Table 2.
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Morphological and structural characterization of bacterial cellulose
Fig. 2 Colony morphology of native Gluconacetobacter hansenii P2A strains and its cellulose non-producing mutants. a Wild type, b celluloseless mutants
Accordingly, cell growth increased proportionally with shaking rate. At 120 rpm, only 2 % of the viable cells were transformed to cellulose-less forms by the end of the fifth cycle, confirming the shear resistance. The increased cell density and the low mutation rate justified for the enhancement in cellulose production. However, at shaking rates of 150 and 200 rpm, mutant colonies appeared just by the first cycle. Since previous batch was used as inoculum for the next batch, mutant ratio increased gradually during consecutive cycles, reaching as high as 64 %, by the end of the fifth cycle at 200 rpm. Mutant colonies were incubated statically in HS medium to check if mutation was reversible. Unfortunately, pellicle formation was never observed, and thus cellulose production ability was not retrievable. Agitated culture conditions were realized at 120 rpm as it minimized the rate of mutant formation. Under the oxygen supply of 0.5 vvm, volumetric mass transfer coefficient (kLa) was calculated as 23 h−1 (data not shown). These conditions were sufficient to attain 30 % of oxygen saturation within the culture medium. By the end of 72 h, the amount of bacterial cellulose synthesized by G. hansenii P2A reached to its highest value, i.e., 3.25±0.11 g/l that corresponded to 0.17± 0.01 yield. Mutant ratio was recorded as 4.0 %, notably higher than shaken culture. The increase in mutant formation was attributed to the combined effect of shear exerted on bacterial cells by the turbine impeller and efficient aeration that led to increased free cell (i.e., not embedded in cellulose) population (Krystynowicz et al. 2002; Park et al. 2003a).
Cultivation conditions affected morphological and structural properties of bacterial cellulose synthesized by G. hansenii P2A. Bacterial cells built a thick biofilm in static culture, while non-uniform cellulose globules were synthesized in agitated and shaken cultures. Morphological structure was studied in detail using SEM images provided in Fig. 3. Accordingly, the cellulosic mat produced under static conditions exhibited an ordered and dense network of fibrils with diameters as thin as 8–10 nm. The cross-sectional view (Fig. 3a) showed that the mat was composed of interconnected layers. On the other hand, synthesized cellulose accumulated in the form of a much looser clump of disordered short and thin fibrils under agitated cultivation conditions (Fig. 3c, d). The difference in aggregation behavior of cellulose fibrils induced by cultivation conditions led to changes in crystallinity as shown by the X-ray diffractograms (Fig. 4a–c). All diffraction profiles showed peaks around 2θ =14.70°, 16.6°, and 22.50° which correspond to typical peaks of 101, 101 , and 002 planes of Cellulose I crystal structure (Ford et al. 2010). Relative crystallinity index of cellulose samples were calculated as 78.7, 72.6, and 77.3 % by Segal method (Eq. 1) for static, shaken, and agitated conditions, respectively. The changes in relative peak intensities of 101 and 101 planes indicated that crystal phase ratios were also different. Mass fractions of crystal phases were calculated using FTIR spectrums that are shown in Fig. 5. The absorbencies of spectral bands at 750 and 710 cm−1 representing Iα and Iβ phases, respectively, were used in calculation of the mass fractions of Iα phase of cellulose samples. FTIR spectrums were also used for description functional groups. The spectral bands of all three bacterial cellulose samples were almost identical and exhibited characteristic bands of Cellulose I structure as reported elsewhere (Bertocchi et al. 1997; Rani et al. 2011; Gea et al. 2011). However, the intensity of spectral band at 895 cm−1 corresponding to β glycosidic linkages (Wong et al. 2009) was suppressed for samples synthesized under agitated conditions. The declines in the proportion of β(1–4) linkages implied that synthesized celluloses had
Table 2 The effect of shaking rate on the conversion of Gluconacetobacter hansenii P2A into non-cellulose-producing mutants Shaking rate
1st Batch
2nd Batch
3rd Batch
4th Batch
(rpm)
cfu/ml
m.r.
cfu/ml
m.r.
cfu/ml
m.r.
cfu/ml
m.r.
cfu/ml
m.r.
0 120 150 200
2.3 × 108 6.2 × 108 1.4 × 109 1.1 × 1010
0 0 0.05 0.10
2.8 × 108 9 × 108 1 × 1010 1.5 × 1010
0 0 0.06 0.15
3.1 × 108 1.9 × 109 1.6 × 1010 2.2 × 1011
0 0.01 0.09 0.24
6.4 × 108 5 × 109 2.3 × 1010 4.7 × 1011
0 0.02 0.10 0.43
7 × 108 1.09 × 1010 3.1 × 1010 6.8 × 1011
0 0.02 0.19 0.64
m.r. mutant ratio calculated as the number of mutant cells divided by the total number of cells
5th Batch
Appl Microbiol Biotechnol Fig. 3 SEM images showing BC produced under stationary culture (a, b) and shaken culture (c, d)
lower molecular weights with respect to the sample synthesized under stationary conditions. The intensity ratios of 750 cm−1 and 710 cm−1 bands were also reduced in agitated synthesis. Through the use of Eq. 2, Iα mass fractions were calculated as 0.88, 0.81, and 0.79 for static, shaken, and agitated cultivation conditions, respectively. Thus, enhanced agitation led to the increase of mass fraction of thermally more stable Iβ crystal phase (Yamamoto et al. 1996). The increase in the mass fraction of Iβ phase suggested a proportional enhancement in thermal stability, which was investigated by thermogravimetric analysis. The thermal behavior of bacterial celluloses synthesized by G. hansenii P2A under different cultivation conditions are shown in Fig. 6. TGA curves of samples produced under static, shaken, and agitated conditions showed single step degradations with maximum decomposition temperatures of 313.3, 320.6, and 332.7 °C, respectively. The thermal stability increased when agitation was applied in culture medium, which confirmed the
gradual increase in Iβ phase content. The residual matter was about 20 % for cellulose produced under static conditions, while 29 % residue was obtained for cellulose produced under shaken conditions. The increase in char ratio was attributed to impurities that also provided a small band in corresponding FTIR spectrum (i.e., at 1,700 cm−1 in Fig. 5c). In the case of agitated culture, the thermal degradation was yet incomplete at 680 °C, which might have aroused from the large sample size with respect to other cellulose samples (6.5 mg vs. 10 mg) or from the substantial increase in decomposition resistance.
Discussion With this study, nine cellulose-producing strains, with production capacities between 0.19 and 4.70 g/l, were isolated from waste and fresh vinegar samples and fruits. Although the recent classification of acetic acid bacteria groups cellulose-
Appl Microbiol Biotechnol
Fig. 5 FTIR spectra of bacterial cellulose samples synthesized in a static culture, b agitated culture, and c shaken culture at 120 rpm by Gluconacetobacter hansenii P2A
Fig. 4 X-ray diffraction spectra of bacterial celluloses synthesized under different cultivation conditions by Gluconacetobacter hansenii P2A: a static culture; b shaken culture; c agitated culture at 120 rpm
secreting strains under Gluconacetobacter genus (Kersters et al. 2006), the existence of several other bacterial species having this phenotypic trait (Czaja et al. 2006) convinced us to carry out morphological and biochemical inspection. Results proved that all isolates complied with Gluconacetobacter genus of Acetobacteraceae family. Even though biochemical testing was not sufficient for identification at species level, interesting and definitive results were obtained for some of the isolates. The most significant result was the liquefaction of gelatin by Gluconacetobacter sp. ESU8. To our knowledge, this was the first report indicating a Gluconacetobacter sp. with positive gelatinase activity. The production capacity of ESU8, i.e., 1.38±0.01 g/l, was similar to the previously reported values for G. hansenii (Park 2003b) and G. xylinus strains (Toyosaki et al. 1995), which implied that this strain could be a corresponding subspecies. However, like all
isolates other than P2A, the cellulose productivity of ESU8 diminished during short-term storage and cell recovery was very poor after resuscitation of freeze stocks. Thus, further research involving 16S rRNA sequencing could not be conducted for these isolates. Gullo et al. (2012) have defined strong relations between subculture frequency and persistence of phenotypic traits, which led us to discuss that 2 weeks of subculturing time was inappropriate for these isolates. Use of cryoprotectants other than glycerol, such as mannitol, malt extract, or sorbitol, might construct another strategy for preservation of viability of freezed cells (Ndoye et al. 2009). These strategies will be implemented in further studies involving isolation of acetic acid bacteria. As a result, isolate P2A was selected as the species with the highest potential for commercial applications owing to its high phenotypic stability during short-term preservation and effective cell recovery after long-term storage, in addition to its sustainable production capacity (Stanbury et al. 1995) of 1.89±0.08 g/l under static cultivation conditions. This value was quite similar to the 2.01 g/l cellulose production by G. hansenii DSM 5602, which was used as reference strain in biochemical testing. The nearly complete 16S rRNA sequence of P2A identified this strain as G. hansenii P2A and thus confirmed the resemblance. Though not a hyperproducer, the production capacity of G. hansenii P2A was found to be significantly higher than the production capacity of G. xylinus K3 (0.217±0.027 g/l) reported by Nguyen et al. (2010) and G. hansenii PJK (0.35 g/l) reported by Park et al. (2003b). Yet, it is possible to enhance production capacity by manipulation of medium composition (Oikawa et al. 1995; Son et al. 2003) or cultivation conditions (Park et al. 2003a; Czaja et al. 2004). Our investigations on cultivation at shaken culture conditions proved that 120 rpm maximized cell growth and cellulose production, while keeping mutation rate at minimum over five consecutive batches. Literature reports on investigation of the effects of shaking rate on bacterial cellulose production by Gluconacetobacter sp. confirm the existence of an optimum shaking rate. Son et al. (2002) have reported that 150 rpm maximized cellulose production by Acetobacter xylinum KJ-1
Appl Microbiol Biotechnol
Fig. 6 TGA curves of bacterial cellulose samples synthesized in static culture (solid line), agitated culture (dotted line), and shaken culture (dashed line) at 120 rpm by Gluconacetobacter hansenii P2A
similarly to Mohite et al. (2012), who have determined that shaking at 200 rpm advances cellulose production by G. hansenii NCIM 2529 strain by 28 %. Though the detected optimum shaking rate in this study was relatively slower, the productivity of G. hansenii P2A was yet very high with respect to other G. hansenii strains reported in literature (Park et al. 2003b), i.e., 2.21±0.03 g/l versus 0.6 g/l, for the same initial medium composition. In contrast to previous literature (Shoda and Sugano 2005), agitation and efficient aeration upgraded the productivity of G. hansenii P2A to 3.25±0.11 g/l, which also emphasized the importance of efficient pH control for this strain. The utilization of supplementary materials such as ethanol and agar were not necessary for the maintenance cellulose producing ability as proposed by Park et al. (2003a) and Bae et al. (2004) indicating the shear resistance of the isolated strain. Culture conditions caused morphological and structural changes in synthesized cellulose by G. hansenii P2A. While significant reductions were observed in fibril dimensions, the crystallinity index of synthesized bacterial cellulose was only slightly affected by shaking and agitation at 120 rpm. The relative crystallinity index of bacterial cellulose can reach as high as 90 %, when bacteria are cultivated under static conditions (Chen et al. 2010; Siró and Plackett 2010), and for a given strain, 5–15 % decline is acceptable for agitated cultivation conditions (Watanabe et al. 1998; Czaja et al. 2004). Bacterial cellulose synthesized by G. hansenii P2A under 120 rpm
agitation exhibited only 1.5 % decline in relative crystallinity index. This was attributed to both shear resistance and attachment of some portion of cells on the agitator and probes, a phenomenon employed in disc bioreactors (Krystynowicz et al. 2002). Shaking and agitation led to 8 % and 10 % increments in Iβ phase ratio, respectively. The increase was accompanied by enhanced thermal resistance with a shift of almost 20 °C in maximum decomposition temperature while the nature of the curve was still typical of pure bacterial cellulose decomposing in a single degradation step that starts usually about 280– 300 °C (George et al. 2005; Gea et al. 2011; Wong et al. 2009). All results proved that G. hansenii P2A could be a candidate microorganism for commercial production of bacterial cellulose. Even though the yield was slightly lower than the 20 % limit (Raspor and Goranovic 2008), further studies are planned for the development of an optimized medium and determination of optimal culture conditions such as pH and temperature aiming for enhancement of the yield and production capacity.
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