Partial bioenergetic characterization of Gluconacetobacter

Partial bioenergetic characterization of Gluconacetobacter

(Parte 1 de 3)

Partial bioenergetic characterization of Gluconacetobacter xylinum cells released from cellulose pellicles by a novel methodology

J.L. Chavez-Pacheco1, S. Martınez-Yee1, M.L. Contreras1,S . Gomez-Manzo1, J. Membrillo-Hernandez2 and J.E. Escamilla1 1Instituto de Fisiologıa Celular, and 2Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico, Mexico

J.L. CHA VEZ-PAC HECO , S. MAR TINE Z-Y E, M.L. CONT RE RAS , S. GO ME Z-MA NZO , J. MEM BRIL LOHE RN A NDEZ AND J.E. ESCAMILLA. 2005.

Aims: Gluconacetobacter xylinum is well known for its ability to produce large amounts of cellulose, however, little is known about its cell physiology. Our goal was to study the respiratory metabolism and components of the respiratory system of this bacterium in static cultures. To reach our goal, a medium formulation had to be designed to improve cell growth and cellulose production together with a novel method for the recovery of cells from cellulose pellicles. Methods and Results: Successive modifications of a nutrient medium improved G. xylinum cell growth 4Æ5-fold under static culture conditions. A blender homogenization procedure for the releasing of cells from the cellulose matrix gave a high yield of cells recovered. Respiratory activities of purified cells were greatly stimulated by exogenous substrates and showed to be resistant to KCN. Unexpectedly, exogenous NADH was oxidized at high rates. Cytochromes a, b, c and d were identified after spectral analyses. Conclusions: Partial bioenergetic characterization of G. xylinum cells allowed us to propose a scheme for its respiratory system. In addition, the growth medium for biomass production and the procedure for the efficient recovery of cells from cellulose pellicles were significantly improved. Significance and Impact of the Study: This work provides the first-ever bioenergetic characterization of G. xylinum grown in static cultures. In addition, a novel methodology to obtain purified cells in suitable quantities for biochemical research is described.

Keywords: bacterial cellulose, bacterial cytochromes, Gluconacetobacter xylinum.

The gram-negative bacterium Gluconacetobacter xylinum is well known for its prolific production of bacterial cellulose, which was first described by Brown (Brown 1886). Bacterial cellulose is endowed with unique physical properties, including a highly crystalline ultrafine fibre network of high purity, therefore is a biological material with many potential industrial uses (for a review see Yamanaka and Watanabe 1994). A major effort has been dedicated to improve biotechnological production of cellulose in cultures (Ross et al. 1991) by testing selected strains (Toyosaki et al. 1995), different fermentor designs (Chao et al. 2001; Cheng et al. 2002; Krystynowicz et al. 2002; Mormino and Bungay 2003), different nutrient formulations (Naritomi et al.

1998a,b; Ramana et al. 2000) and by modifying partial O2 and CO2 pressures (Kouda et al. 1997; Hwang et al. 1999).

Correspondence to: J.E. Escamilla, Instituto de Fisiologıa Celular, Universidad Nacional Autonoma de Mexico. Apdo. Postal 70-242 Mexico City 04510 (e-mail: eescami@ifc.unam.mx).

ª 2005 The Society for Applied Microbiology

Journal of Applied Microbiology 2005, 9, 1130–1140 doi:10.1/j.1365-2672.2005.02708.x

Despite the fact that O2 availability in G. xylinum cell cultures has been recognized as a crucial factor for the production of cellulose, the study of the bioenergetic properties of this bacterium has been overlooked mainly due to the lack of a proper procedure to obtain cells from the cellulose matrix (Watanabe and Yamanaka 1995; Verschuren et al. 2000). Remarkably, notwithstanding this limitation, Benziman and Galanter (1964) carried out seminal experiments on the partial purification and characterization of the respiratory chain-linked malate dehydrogenase. Later on, the same group identified Ubiquinone10 as constituent and functional part of the respiratory system and provided a tentative composition of membrane cytochromes (Benziman and Goldhamer 1968). More recently, Matsuoka et al. (1996) reported on the respiratory activities associated to membranes and determined the H+:O2 ratios evoked by several substrates in intact cells. It is important to note that all the above studies were performed in cells obtained from shaking cultures, a growth condition where cellulose is made as an irregular-shape product with low value for biotechnological applications.

In static cultures, G. xylinum cells are confined within the growing cellulose network and remain firmly attached after harvesting. To free cells from cellulose pellicles, hand squeezing or gentle mechanical treatments have been used to prepare limited cell quantities for further studies (Swissa et al. 1980; Hwang et al. 1999; Son et al. 2001; Heo and Son 2002); alternatively, cellulose accumulation has been counteracted by the addition of commercial cellulase to cultures (Matsuoka et al. 1996); in both cases, low cellular yields are obtained therefore hampering biochemical studies where high quantities of biomass are required.

In this communication, a partial bioenergetic characterization of whole cells of G. xylinum was carried out to get insight into the respiratory metabolism and into the nature of molecular components of its respiratory system. At the same time, culture conditions and the cell release procedure to obtain higher cells yield were improved.

MATERIALS AND METHO DS Organism and culture conditions

Storage conditions. Gluconacetobacter xylinum IFO 13693 was kept at )70 C in a basal-modified medium (BM- medium) plus glycerol, containing (g l)1): sucrose, 50;

The BM-medium has the same components of the WY- medium reported earlier (Watanabe and Yamanaka 1995) except that our BM-medium does not contain ammonium sulfate.

Culture conditions. The strain was activated in 250 ml Erlenmeyer flasks containing 100 ml of BM-medium, incubated at 30 C for 48 h at 150 rev min)1. To optimize cell growth yields in static cultures, the BM-medium was modified as indicated in Fig. 1. Static 500 ml cultures were grown in 2 l Fernbach flasks and incubated for 10 days at 30 C. Ethanol (1Æ4% v/v), sugar (5% w/v) and nitrogen sources (1% w/v) were added as indicated in Fig. 1 legend. To verify the buffer capacity of the media, potassium phosphate concentration was changed in the ranges of 20–200 mmol l)1, initial pH was adjusted to 6Æ0 with NaOH.

Preparation of isolated cells from cellulose pellicles

Cell recovery. Pellicles [800 g wet weight (w)] were harvested after 10 days of growth on square aluminium trays (45 · 35 · 8 cm) containing 1 l of the selected culture medium. Pellicles were suspended in 2 l of potassium phosphate 100 mmol l)1,p H 6Æ0 and homogenized (six cycles of 30 s, resting on crushed ice for 5 min between cycles) in an industrial blender (5 l jar) at 4 C. The resulting suspension was filtered through a piece of felt mesh (commercial fabric) and centrifuged at 8670 ·g for 10 min. Filter-retained cellulose was subjected to a second round of blending. Cell pellets were mixed, washed twice with same phosphate buffer and quantified as w. In some experiments, the homogenization buffer was supplemented with NaCl (0Æ1 mol l)1) or Tween 20 (0Æ1%) in order to promote cell release. Cellulose residues were boiled in NaOH 0Æ5 mol l)1 for 30 min, thoroughly washed with distilled water and quantified as dry weight (dw) after 5 h at 80 C.

Electron microscopy analysis. Cellulose and cells were fixed in buffered glutaraldehyde 2Æ5% followed by osmium tetroxide 1%. Fixed samples were washed once with potassium phosphate 0Æ1 mol l)1,p H 7Æ0 and dehydrated by the addition of alcohol (from 30 to 100%). Preparations were dried to critical point and gold stain was carried out before visualization in a JEOL JSM- 5410LV scanning microscope (Molinari et al. 1998).

Respiratory activities and spectral analyses

Oxidase activities. Oxidase activities at 30 C in whole cells were determined with a Clark oxygen electrode using a 53YSI oxygenmeter as previously described (Contreras- Zentella et al. 2003). Briefly, cells were suspended in a final volume of 2 ml of potassium phosphate buffer (100 mmol l)1 pH 6Æ0o r7 Æ4). Respiratory substrates were added at 10 mmol l)1 (glucose, ethanol or acetaldehyde) with the exception of NADH (5 mmol l)1). The reaction

G. XYLINUM RESPIRATORY SYSTEM 1131 ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 9, 1130–1140, doi:10.1/j.1365-2672.2005.02708.x

was initiated by the addition of 50–100 l of cel suspension (0Æ1g w ml)1). The low endogenous-substrate respiration was subtracted from the activity evoked by each of the added substrates. Inhibition kinetics using potassium cyanide (up to 4 mmol l)1) was determined on the oxidase activity with acetaldehyde as substrate.

Spectral analyses. Gluconacetobacter xylinum cells were suspended in potassium phosphate buffer (50 mmol l)1, pH 6Æ0), supplemented with dimethylsulfoxide (DMSO; 25% v/v) or polyethylenglycol 3350 (PEG 3350; 25% v/v) to obtain homogeneous freezing of samples. Spectra at 7 K were recorded in an Olis DW2000 spectrophotometer using 2 m light path cuvettes. Samples were reduced with a small quantity of solid sodium dithionite, NADH (5 mmol l)1) or any of the substrates (10 mmol l)1) as indicated in the figure legends (Figs 4 and 5). References were oxidized with a small quantity of ammonium persulfate. To obtain the CO-difference spectrum, both cuvette compartments were reduced with glucose, and then, the sample cuvette was bubbled for 5 min with a gently stream of CO, before freezing (Flores-Encarnacion et al. 1999). To obtain the photodissociation difference spectrum of heme-CO compounds, samples reduced with glucose and bubbled with CO were scanned at 7 K to obtain the prephotolysis spectrum; then, the frozen sample was photolysed with three close shoots of a Vivitar V2000 electronic flash and the postphotolysis spectrum was recorded. The photodissociation difference spectrum of heme-CO compounds was obtained by subtraction of the prephotolysis spectrum from the postphotolysis spectrum (Kelly et al. 1993).

Optimization of a growth medium for Gluconacetobacter xylinum

Conditions for cell growth in static cultures were significantly improved by a successive modification of the BM- medium (see Materials and methods). After 10 days of cultivation, 0Æ28 g of cells (w) and 0Æ81 g of cellulose (dw) were produced per litre of BM-medium (Fig. 1a). Addition of 1Æ4% (v/v) ethanol to the BM-medium increased cell yield (about 1Æ9-fold) with a marginal increase in cellulose production (about 1Æ1-fold, Fig. 1a). These results are in good agreement with previous reports showing that ethanol improves growth and cellulose synthesis in G. xylinum (Matsuoka et al. 1996; Naritomi et al. 1998a; Krystynowicz et al. 2002). Thus, ethanol was incorporated as a component of the different media tested. Replacement of the original sucrose by glucose further increased cell growth and cellulose production (about 1Æ5-fold in both cases; Fig. 1b). The addition of other sugars such as fructose, sorbitol or lactose instead of sucrose did not improve cell growth nor cellulose production (Fig. 1b). An increase in buffer capacity by changing the potassium phosphate concentration from 20–200 mmol l)1 had little impact on growth or cellulose production (Fig. 1c); noteworthy, highest growth : cellulose ratio was observed at 100 mmol l)1 phosphate. The final pH of cultures was slightly below 3Æ0 when either 20 or 50 mmol l)1 potassium phosphate was used and slightly above 3Æ0 when 100 or 200 mmol l)1 phosphate was used. Finally, in addition to the yeast extract present in the BM-medium, other nitrogen sources such as glutamate, ammonium sulfate, casein hydrolysate and grenetin (animal protein), were tested (Fig. 1d). Under

(g l

Ba Et Gl Fr So La 20 50 100 200 Glu Cas Gre Am Sugar Phosphate Nitrogen

Fig. 1 Culture medium optimization for cell growth (solid bars, g l)1 w) and cellulose production (empty bars, g l)1 dw) by G. xylinum IFO 13693 in static cultures. BM- medium containing 5% (w/v) sucrose and 0Æ5% (w/v) yeast extract as carbon and nitrogen sources was modified to improve cell growth. (a) Effect of 1Æ4% (v/v) ethanol. (b) Effect of different sugar sources added at 5% (w/v). (c) Effect of different phosphate concentrations (mmol l)1). (d) Effect of different nitrogen sources added at 1% (w/v). Abbreviations: Ba, basal; Et, ethanol; Gl, glucose; Fr, fructose; So, sorbitol; La, lactose; Glu, sodium glutamate; Cas, casein hydrolysate; Gre, grenetin; Am, ammonium sulfate

1132 J.L. CHA VE Z- PACHECO ET AL.

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 9, 1130–1140, doi:10.1/j.1365-2672.2005.02708.x our experimental conditions, grenetin did not improve cell growth. By contrast, sodium glutamate increased cell growth by about 1Æ5-fold compared with that of the BM-medium supplemented with ethanol plus glucose (compare Fig. 1c,d). Casein hydrolysate and ammonium sulfate were less effective (about 1Æ2 and 1Æ1-fold, respectively). Our results are consistent with a recent report where sodium glutamate, among other compounds, was identified as a good nitrogen source for G. xylinum static cultures (Ramana et al. 2000). Taken together, these results indicate that the modifications made to the BM-medium (addition of ethanol, glucose instead of sucrose, sodium glutamate and 100 mmol l)1 phosphate) favour cell growth (about 4Æ5-fold higher) and cellulose production (only 1Æ8-fold higher) in static cultures. Our final medium formula, here called BEGG-medium contains (g l)1): glucose, 50; yeast extract,

Gluconacetobacter xylinum cell recovery from cellulose pellicles

A reliable method for the recovery of G. xylinum cells trapped in cellulose pellicles has been addressed here. The hand-squeezing of pellicles is the standard procedure used to obtain low amounts of cells (Hwang et al. 1999; Son et al. 2001). Moreover, cellulose accumulation has been counteracted by the costly addition of cellulase (Matsuoka et al. 1996). Thus, it was decided to look for a more simple and reliable method. Preliminary attempts showed that mechanical homogenization of pellicles in a home blender liberated significant amounts of cells; therefore, it was decided to scale up the procedure in a commercial blender (3 l working volume in a 5 l jar) using six 30 s homogenization cycles with intervals of 5 min, in an ice bath between cycles. Thereafter, the homogenizate was filtered through a felt mesh. The filtrate thus obtained was centrifuged for 10 min, at 8670 ·g. Pelleted cells were washed twice with 100 mmol l)1 potassium phosphate buffer (pH 6Æ0). The use of blender homogenization showed a significant increase in cell recovery (about twofold; Table 1).

Scanning electron microscopy of cellulose pellicles before and after the blending treatment (Fig. 2a,b, respectively) showed that significant amounts of released cells form aggregates (Fig. 2b), suggesting that cell aggregates were retained together with cellulose particles by the felt mesh. In order to prevent cell aggregation, the blender homogenization step was either performed in the presence of a gently detergent (0Æ1% Tween 20, Fig. 2c) or salt (0Æ1 mol l)1 NaCl, Fig. 2d). In both cases, cell cluster dispersion was evident. The addition of Tween 20 or NaCl increased biomass recovery about 2Æ5–3Æ6-fold, respectively when compared with the hand squeeze protocol (Table 1). It is important to note that cells recovered after blender homogenization were apparently intact but still moderately contaminated with cellulose debris that could not be washed out by centrifugation (Fig. 2e).

Respiratory activities of Gluconacetobacter xylinum purified cells

Physiological studies of cells released by hand-squeeze or blender homogenization were compared by determining respiration activities of periplasmic PQQ-dehydrogenases and membrane-bound NADH dehydrogenase evoked by different substrates, such as glucose, ethanol or acetaldehyde. As shown in Table 1, cells obtained by handsqueezing, as the most gentle procedure, displayed the highest activities, close followed by cells recovered after blender homogenization in buffer alone and buffer plus NaCl. Respiratory activities of cells released in buffer plus Tween 20 were consistently slightly lower. Surprisingly, exogenous NADH evoked high respiratory activities in cells released by blending. As NADH oxidation by Complex I (NADH dehydrogenase) takes place at the inner side of the cytoplasmic membrane and exogenous NADH usually does not permeate the membrane (Overkamp et al. 2000), membrane damage caused by the blender treatment was suspected; however, cells released by the gently handsqueeze treatment showed similar NADH oxidation activities (Table 1). Respiration evoked by exogenous NADH in whole cells was previously reported for Methylophilus methylotrophus, however it was later shown to be an artefact

Table 1 Biomass yields and substrate stimulated respiration of G. xylinum cells released from cellulose pellicles Treatment Biomass (g wet cells l)1)

O2 uptake (nmol O2 min)1 mg)1 wet cells) Acetaldehyde Ethanol Glucose NADH

G. XYLINUM RESPIRATORY SYSTEM 13

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 9, 1130–1140, doi:10.1/j.1365-2672.2005.02708.x

(a) (b)

(c)(e) (d)

Fig. 2 Scanning electron microscopy of cellulose pellicles and cells purified from G. xylinum IFO 13693. (a) Untreated cellulose pellicles obtained after 10 days of static culture. (b) Cellulose residues obtained after blending treatment in 100 mmol l)1 phosphate buffer, pH 6Æ0. (c) Cellulose residues after blending treatment in the presence of 0Æ1% Tween 20 or (d) in the presence of 0Æ1 mol l)1 NaCl. (e) Cells purified after blending treatment in the presence of NaCl. The bar is equivalent to 1 lm for all the micrographs with the exception of panel b where the bar equals to 5 lm

1134 J.L. CHA VE Z- PACHECO ET AL.

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 9, 1130–1140, doi:10.1/j.1365-2672.2005.02708.x caused by the oxidation of residual ethanol present in the NADH commercial preparations (Patchett and Jones 1986). Such an artefact in G. xylinum cells was discarded, measuring the exogenous NADH oxidation spectroscopically at 340 nm, a condition that avoids interference caused by the parallel oxidation of contaminant ethanol. Spectroscopically determined NADH oxidase activity (52 nmol of NADH min)1 mg)1 cell w) was somewhat lower than the activity determined with the Clark oxygen electrode (Table 1) yet, shows that a significant part (78%) of the activity amperometrically determined is actually due to NADH-oxidase.

Gluconacetobacter xylinum respiration evoked by acetaldehyde was inhibited with different KCN concentrations in cells grown in BEGG or BEGG without ethanol medium (Fig. 3). The absence of ethanol during growth led to 50% decrease in the activity levels of ethanol oxidase but without detectable changes in other oxidase activities (data not shown). Regardless of the presence of ethanol in the cultures, KCN inhibition of cell respiration showed biphasic kinetics, the KCN-sensitive component apparently contributing more to total respiration in those cells grown without ethanol (50 lmol l)1 KCN caused 10 and 30% inhibition in cells grown with and without ethanol, respectively; see inset in Fig. 3). Similar cyanide inhibition kinetics were obtained for the other substrates (data not shown). Thus, the cyanide-resistant pathway seems to be dominant in G. xylinum cells grown in static cultures and its contribution to the total respiration increased when ethanol is present as energy source (Fig. 3).

Cytochrome content of the respiratory chain of Gluconacetobacter xylinum

In order to determine the cytochrome respiratory components of G. xylinum, cells harvested using our novel method were subjected to spectral analyses to obtain the reduced difference spectra using different substrates as reducing agents (Fig. 4a). Whole cell samples were initially suspended in buffer containing 25% DMSO, however we noted that ethanol oxidation by whole cells was impaired by this solvent concentration (data not shown). Therefore, the spectrum evoked by ethanol (Fig. 4a, trace 1) showed poor reduction levels. By contrast, dithionite, NADH, acetaldehyde and glucose produced stronger signal profiles and reduction levels (Fig. 4a, traces 2 to 5). Thus, exogenous NADH caused efficient cytochrome reduction (Fig. 4a, trace 3) in good agreement with the high O2-uptake observed when exogenous NADH was used as substrate (Table 1), sug- gesting that NADH oxidation by whole cells could be a distinctive property of G. xylinum. However, an artefact caused by NADH permeation in damaged cells can not be ruled out.

Reduction by dithionite or physiological substrates

(Parte 1 de 3)

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