Microbial carbon-substrate utilization in the rhizosphere of Gutierrezia sarothrae grown in elevated atmospheric carbon dioxide

Soil Bid. Biochm. Vol. 29, No. 9-10. pp. 1387-1394, 1997 0 1997 Elsevier Science Ltd. All rights resewed Printed in Great Britain PII: soo38-0717(97)ooos3-9 0038-0717/97 517.00 + 0.00

Pergamon

MICROBIAL CARBON-SUBSTRATE RHIZOSPHERE OF GUTIERREZIA IN ELEVATED ATMOSPHERIC MATTHIAS

UTILIZATION

IN THE GROWN CARBON DIOXIDE SAROTHRAE

C. RILLIG,‘* KATE M. SCOW; JOHN N. KLIRONOMOS’ and MICHAEL F. ALLEN

‘Department of Biology, Soil Ecology and Restoration Group, San Diego State University, San Diego, CA 92182, U.S.A., *Department of Land, Air and Water Resources, University of California, Davis, CA 95616, U.S.A. and ‘Department of Botany, University of Guelph. Guelph, Ontario, Canada NIG 2Wl (Accepted

24 January 1997)

Summary-Differences in rhizosphere microbial community function in response to Gutierreziu plants grown in elevated CO2 (750 ~1 I-‘) and fertilized with nitrogen were studied using the

sarothrae

Biolog microplate analysis of sole C substrate utilization. Compared to ambient CO*. under elevated CO*, polymers were more slowly oxidized by the microbial community, amides showed no change in usage, and all other substrate groups were more rapidly utilized, although there was no significant change in the number of viable bacteria. No microbial community responses to N fertilization were detected. The results indicate that potential functional changes in the soil microbial community in response to elevated CO2 have to be taken into account in future experiments. Differential use of rhizodeposits in elevated CO2 may have important consequences for biogeochemistry and plant growth. Q 1997 Elsevier Science Ltd

INTRODUCTION Measurements of atmospheric CO2 concentrations and of air bubbles trapped within ice caps indicate that global CO2 concentrations have been rising for several decades after thousands of years of relatively constant concentrations (Houghton et al., 1990; Keeling et al., 1995). The direct physiological effects of elevated carbon dioxide on plants have been well studied (Bowes, 1993). The main effects are increased photosynthetic rate, increased wateruse efficiency, and higher translocation of photosynthate to the root and rhizosphere. Experiments with elevated CO2 at the ecosystem level have indicated that a great proportion of the increase in fixed carbon is allocated below ground (Kiirner and Arnone, 1991; Stocker et al., 1995). Since a major part of net primary productivity of a variety of ecosystems is channeled below ground (Fogel, 1985; Schlesinger, 1991), the fate and path of this belowground C are important to global change biology and need to be determined. On a larger spatial and temporal scale, it is not known whether C will be sequestered in slow-turnover pools in the soil or rapidly mineralized and hence fed back to the atmosphere. By providing a C source for rhizosphere microorganisms, that C also may affect the heterotrophic microbial community. *Author for correspondence. SFJB29/9-10--D

We need to understand better how potential changes in the soil microbial community will feed back on nutrient cycles and hence plant growth. Carbon can reach the soil compartment of ecosystems via three routes: rhizodeposition, belowground litter and above-ground litter production. There have been studies on decomposition of above-ground litter produced in elevated CO2 compared to ambient (CoQteaux et al., 1991; Kemp et al., 1994; O’Neill and Norby, 1996). The evidence is inconclusive, but it is conceivable that rates of decay of above-ground litter will be unaffected by elevated CO2 (O’Neill and Norby, 1996). Gorissen et al. (1995) were the first to describe how belowground litter decomposition is changed by elevated CO*. They found that after an initial period of rapid decomposition, there was a substantial delay in litter decomposition. Rhizodeposition is difficult to measure directly in situ because of problems of collecting the rhizodeposit in the soil environment. However, rhizodeposition is expected to increase under elevated COz (Rogers et al., 1994). Increases in rhizodeposition may give rise to a larger microbial biomass, since soil microbes are typically carbon-limited (Paul and Clark, 1989). This larger microbial biomass will affect plant nutrition. There are currently two hypotheses regarding this effect on plant growth, depending on whether nutrient immobilization (Diaz et al., 1993) or mineralization (Zak et al., 1993) is the more dominant

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Matthias C. Rillig et al.

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process. In the case of nutrient immobilization, nutrients are immobilized into microbial biomass and are hence made unavailable to the plant, at least temporarily; the effect on plant growth would be negative. In the case of mineralization, nutrients will become increasingly available to plants, resulting in positive effects. However, in both models, soil microbes are treated as one “black box” of soil microbial biomass, thereby ignoring the functional diversity of soil microbes. Studies on rhizosphere microbial responses to plants grown in elevated CO1 have been largely limited to specific groups of organisms. These include nitrogen-fixing bacteria 1995), mycorrhizal fungi (Vogel and Curtis, (O’Neill, 1994; Klironomos et al., 1996) and nonmycorrhizal fungi (Klironomos et al., 1996). We have used Biolog (Biolog, Inc., Hayward, CA, U.S.A.) microplates as a tool for comparing microbial carbon utilization patterns in soil in response to Gutierrezia sarothrae grown under elevated atmospheric CO*. Our main objectives were (a) to link putative changes in the use of specific substrates to elevated CO*, and (b) to correlate potential shifts in utilization of C substrate groups with CO?. We also examined the effects of N fertilization, since soil nutrient status may be an important modifier of COz effects. METHODS

Plant material (Gutierrezia sarothrae snakeweed Broom Shinners.) is a common, woody, short-lived perennial that occupies 60% of some southwestern U.S. rangelands (Wan et al., 1993). Forty 3-y-old plants were harvested in July 1994 from a recolonized burnt site at San Diego State University’s Sky Oaks Biological Field Station in San Diego County, California (33”23’N, 116”37’W; 1300 m elevation and 75 km from the coast). Soil from the site (for a characterization see Allen et al., 1996) was excavated to 25 cm depth, mixed and sieved (1 cm). The plants were transplanted individually into 50-cm-tall pots (15 cm dia) immediately after harvest. We used a 2 x 2 factorial experimental design, with the two factors being atmospheric COz (ambient vs elevated) and soil N (fertilized vs non-fertilized). Forty plants were grown for 4 months in four Conviron (Controlled Environments, Ltd, Canada) PGRl5 growth chambers at the University of California Davis Controlled Environment Facility. The growth conditions were set as follows: photoperiod (light: dark) 12: 12 h; photosynthetic photon flux density at canopy height was 700 pm01 mm2s-‘; temperature 23: 13°C (day/night); relative humidity 60%/95% (day/night). Two chambers were set at 750 ~1 COz 1-l (elevated), the other two were running at 350 ~1 CO* 1-l (ambient). In each chamber, half the pots received N-fertiliza-

tion. Fertilization consisted of a CaNOs plus NHdNOs (nitrate: ammonium, 2: 1) mixture (equivalent to 100 kg ha-‘). Water in the N treatment contained the appropriate amount of fertilizer. Pots were watered to field capacity at the beginning of the experiment, and then with 200ml of water every week, gradually reducing the soil moisture content to 3.8% (w/w; SE = 0.2%). This simulated the transition from growing season to dry season conditions prevalent in the semi-arid climate of southern California chaparral. At the end of the experiment, plant fresh and dry (after drying in 65°C for 3 d) weights were measured. Shoots were clipped off at the base, and roots were obtained by wetsieving after samples for Biolog analysis were taken. Soil samples for microbial analysis were taken at a depth of approximately 15 cm, instantly cooled on ice and stored in the dark until analysis. Rhizosphere soil could not be separated from bulk soil because of the low soil moisture at harvest time. However, samples were always taken in regions with a high fine root density. Biolog analysis

Microbial carbon-use was analyzed using Gramnegative (GN) microplates (Biolog, Inc.). Each of the 96-well GN-microplates contains one of 95 substrates in a buffered nutrient medium (plus a control well) and a tetrazolium dye (Garland and Mills, 1991). The degree to which each of the substrates is being oxidized by the mixture of bacteria used as inoculum is assayed by calorimetrically measuring tetrazolium dye reduction. All soil samples were processed for use with Biolog plates within 5 d of harvest. One gram of soil (dry weight), including three intact l-cm-long root fragments were used for micorbial plate inoculum. Soil microbes were suspended by shaking the samples in 50 mM phosphate buffer (prepared with cell-free autoclaved water) for 5 min. Because the soil was very sandy with a low amount of organic matter, no centrifugation step was included. Each well received 140 ~1 of suspension, applied with a multipipettor. A test had showed that there was a 40-h lag in plate well color development, and that a dilution of lop2 produced the most consistent carbonuse patterns on three replicate plates. Including root fragments did not cause color development in control wells (no carbon substrate) in any of the plates used in this experiment. For each pot, two replicate extractions were carried out for a total of 80 Biolog plates. Incubation temperature was 25°C. At 48, 72, 96 and 120 h after inoculation, absorptions in Biolog-plate wells were read at 595 nm with a microplate reader (Molecular Devices, Inc. Crawley, U.K.) equipped with SOFTmax software (version 2.01). Absorbance values for the wells with C sources were blanked against the control well, yielding corrected 0Dsg5 values. Overall color

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Elevated CO2 and soil microbial C utilization

development in a Biolog plate was expressed as average well color development (AWCD) according to Garland and Mills (1991). Bacterial numbers

A differential fluorescent staining (DFS) procedure was used to measure viable bacterial numbers as described in Conners et al. (1994). The DFS was prepared by mixing the chelate, europium (III) thenoyltrifluoroacetonate, with a fluorescent brightener (Anderson and Westmoreland, 1971). Dilutions were used to make smears, which were subsequently covered with DFS for 1 h. The samples were then rinsed with a 50% ethanol wash. Stained slides were then viewed under UV (620 nm), where active cells were detected as red fluorescence. Tests had suggested that this DFS method stains living bacterial cells, making it a good indicator of (S. Morris, T. Zink, J. microbial activity Klironomos, and M. F. Allen, in preparation). Statistical analvsis Two-way analysis of variance (ANOVA) was used to analyze the plant and bacterial biomass data. Residuals were checked for normality with ShapirooWilks’ W-test and for homogeneity of variance with Levene’s test. Microbial carbon utilization was analyzed using Principal Component Analysis (PCA) performed on the correlation matrix using the computer program CANOCO (Ter Braak, 1990). PCA is a useful tool for analyzing data sets with a large number of response variables (here: C substrate oxidation), and has been employed several times to explain multivariate Biolog data sets (Zak et al., 1994). In adCanonical dition, we also performed a Correspondence Analysis (CCA), using the same statistical software package. This method has the advantage that the axes are constrained to be linear combinations of environmental variables (i.e. CO2 and N), rather than linear combinations of response variables. This ordination method allows for the calculation of a P-value for the environmental variables. P-values for environmental variables in CCA were obtained by a Monte Carlo permutation test (999 permutations). For either ordination method, the two replicates for each pot were averaged. Samples from pots that had excessively low bacterial counts, and from pots where the plant died during the experiment were not included in the analysis (four samples removed). Comparisons between C substrate oxidation in ambient and elevated CO* were made for 10 substrate groups separately, using 10 t-tests (df = 34) per reading time. For each substrate group and pot, for example phosphorylated compounds, the average oxidation value was obtained by summing the 0Ds9s values for all phosphorylated compounds and then diving by their number.

RESULTS Plant-growth responses Gutierrezia sarothrae clearly responded to the elevated CO2 and the nitrogen treatment (Fig. 1). Root dry weight increased by 33% in response to elevated CO* (P = 0.009) and by 35% to the N fertilization (P = 0.008). The interaction term was not significant (P = 0.73). Shoot dry weight did not change significantly for either CO2 (P = 0.44) N (P = 0.94) or the interaction (P = 0.18). Shoot fresh weight, however, increased significantly under elevated CO* (P = 0.003), as did root fresh weight in response to elevated CO* (P = 0.0001) and to added N (P = 0.0009). The CO* x N interaction term was not significant for root (P = 0.39) or shoot (P = 0.47) fresh weight. Number of viable bacteria

The number of bacteria did not change significantly with either elevated CO2 (F = 1.42, P = 0.24) added N (F = 0.05, P = 0.82) or CO* x N (F = 0.011, P = 0.92) (Fig. 2).

(a) 15

DRY WRIGHT

Control

Cq

N

CqXN

(b)

I FRRSHWRIGHT

Coniml

C&

CqXN

Fig. I. Effects of elevated CO2 and N addition on plant root and shoot dry weight (a) and fresh weight (b). Error bars are standard errors of the mean.

Matthias C. Rillig et al.

Control

Co;!

N

CqXN

Fig. 2. Effects of elevated CO* and N addition on the number of bacteria g-’ soil. Error bars are standard errors of the mean.

Biolog analysis

Soil samples separated along the first principal component (PC) axis for all three reading times (Fig. 3). There was no apparent pattern with respect to the N treatment. The first PC axis explained 32% of the variation for the 72 h reading, 25% for the 96 h reading, and 22% for the 120 h reading. Eigenvalues for the second PC were much lower: 9.5% (72 h), 7.6% (96 h), and 10.3% (120 h). There was no discernible separation of samples along the second axis. Although there are various stopping rules for the best number of principal components (Jackson, 1993), we restricted our discussion to PC 1, which provided the separation of interest in our data set, and which had by far the highest eigenvalue. The PCA findings were corroborated by canonical correspondence analysis. COz was a significant environmental variable (72 h reading: P = 0,001; 96 h reading: P = 0.001). whereas nitrogen was not (72 h reading: P = 0.4; 96 h reading: P = 0.6). The interaction term was significant (72 h reading: P = 0.03; 96 h reading: P = 0.003). The separation of samples along the first PC axis can be related to differences in sole C source oxidation by examining the correlation of the response variables with this axis. To do this, substrates that showed a consistent trend across all reading times were identified. Those carbon sources were then grouped into three response types: positive correlation with PC axis 1 (r > OS), negative correlation with PC axis 1 (r < -OS), and no correlation with is being used substrate PC axis 1, but (-0.2 < r < 0.2). Twenty-four such substrates, or 25% of all C sources, showed a consistent trend and could be grouped in this way (Table 1). The other substrates showed no consistent trends; this behavior has been observed before (Bossio and Scow, 1995). Most C sources showed a positive cor-

relation with PC axis I, i.e. they became increasingly or more rapidly oxidized in elevated COz than in ambient COz. This trend is supported by examining the mean AWCD of plates across the treatments (Fig. 4), which showed an increase along the COz gradient. Nitrogen additions had no effect on AWCD for any reading time or concentration of atmospheric CO2 (Fig. 4). AWCD can be regarded as an indicator of microbial activity of the culturable microbes as they use the substrates provided in the microplates. However, there were also substrates that were used less rapidly in elevated CO*, including an ester (methyl pyruvate) and three carboxylic acids (formic acid, quinic acid, succinic acid). Four substrates showed no change in usage, despite the fact that they were being used, in some cases quite strongly. These included two carbohydrates (i-erithritol and D-psicose), a carboxylic acid (propionic acid), and an amide (succinamic acid). Substrates were grouped into 10 groups according to their chemical structure. When well color development was averaged over those groups, patterns in utilization emerged that were consistent over all reading times (Fig. 5). Color development in wells containing phosphorylated compounds showed the most rapid increase in the elevated COz treatment. amino Alcohols, amines, aromatic compounds, acids and carbohydrates each increased significantly as a group as well. Oxidation of amides did not change significantly, and carboxylic acids showed no consistent trends. Polymers and esters show a decreased utilization for the 120-h and 96-h readings, respectively. Individual polymer compounds did not show consistent trends; this pattern was only apparent at the substance group level.

DISCUSSION It is clear from our study that qualitative changes in the rhizosphere microbial community may occur in response to elevated atmospheric COZ, irrespective of a change in microbial biomass. Substrate utilization by the microbial community associated with Gutierrezia sarothrae roots changed significantly with elevated atmospheric COz, although there was no significant change in numbers of viable bacteria. These changes in microbial carbon-utilization patterns suggest that there were changes in rhizodeposition under elevated COz. At this point, we are not certain as to whether there was any change in the total amount of rhizodeposit, in the composition of the rhizodeposit, or both. However, direct effects of elevated CO* on soil organisms have, in general, been discounted (Van Veen et al., 1991). We cannot exclude that below-ground litter production (dead roots) caused part of the difference in C utilization. Above-ground litter can be excluded as a cause, since there was no litter accumulation.

Elevated CO* and soil microbial C utilization (a) 72

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h reading

EIevated CO2 I

NON

??

With N

Ambient CO2

(b) 96 h reading

0

NON

0

With N

+

ORIGIN

(c) 120 h reading

Fig. 3. Ordination diagram produced from Principal Component Analysis of samples from pots of the different treatment combinations (symbol types). (a)-(c) are the results obtained by measuring at different reading times. Scores of each sample for the first and second principal component (PC) are plotted.

A stratified sampling approach can be used in further experiments, distinguishing between the microbial communities associated with dead or live roots. Interestingly, microbial function was modified in response to elevated CO2 without any changes in total numbers of viable bacteria. Such a shift in microbial function likely altered nutrient cycling and feedbacks to plant growth, since rhizosphere microbes influence resource availability to the plant (Curl and Truelove, 1986: pp. 87-90; Diaz et al., 1993; Zak et al., 1993). Polymers were less rapidly oxidized by the microbial community under elevated COz. This could be explained by three alterna-

tive hypotheses. First, fewer polymeric compounds were contained in the rhizodeposit of Gutierreziu exposed to elevated CO* (relative shift in rhizodeposition). Second, the actual amount of polymer rhizodeposition did not change, but microbes preferentially utilized other compounds, such as simple carbohydrates, whose concentration in the rhizodeposit may have increased, over polymers (utilization shifts). Third, members of the bacterial community that degrade polymers were outcompeted by other members of the community in the altered resource environment (community interactions). From the point of view of the microbial community composition, two hypotheses can be

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Matthias C. Rillig et al.

Table I. Single carbon source variables. trate groups. that showed a consistent

axis

sorted according to susbtrend in correlation with PC

I overall reading times

Substrates with positive correlation (r>O.5) with PC1 Carbohydrates

Carboxylic acids Amino acids

Aromatic compounds Amines Alcohols Phosphorylated compounds Substrates with negative correlation (r < -0.5) with PC1 Esters Carhoxylic acids

t-Arabinose m-Inositol D-Trehalose Turanose g-Hydroxybutyric acid p-Hydroxy phenylacetic acid D-Alanine t-Alanyl-glycine Hydroxy t-proline L-Proline t-Pyroglutamic acid L-Serine Uridine Putrescine Glycerol Glucose-l-phosphate

Methyl pyruvate Formic acid Quinic acid Succinic acid

Substrates with no correlation (-0.2 < m < 0.2) with PC1 Carbohydrates

i-Erythritol D-PSiCOSe

Carboxylic Amides

acids

Propionic acid Succinamic acid

given: either the species composition was changed in elevated CO*, or microbes have differentially adapted to substrates, without any changes in the microbial community. These hypotheses are not mutually exclusive. They are important hypotheses to pursue in future experiments, since a reduced oxidation of polymers could lead to an increased build-up of recalcitrant organic matter, and eventually increased C sequestration in the soil. The equivalent hypotheses for a mechanistic explanation of the considerable increase in the usage of phosphorylated compounds can be given as outlined for the decrease in polymer utilization. The observation of increased oxidation of these compounds also may be important in describing the effect of the soil community on plant growth. Rapid mineralization of phosphorus will be beneficial to plant growth, if the soil community does not outcompete plant roots (Diaz et al., 1993). Current conceptual models of the soil microbial response to elevated COz (Diaz et al., 1993; Zak et al., 1993) do not include the possibility of changes in C utilization or community composition, but are instead focused on total microbial biomass. This contains the implicit assumption that the different soil microbes will increase proportionately under elevated COz, without any changes in composition. Our results show that this may be an oversimplification. Total microbial biomass cannot detect poten-

I 48

, 72

+

CONTROL

-

CO2

__-o-_

N

--A----

CO2 X N

96

120

Plate reading time (h)

Fig. 4. Color development (AWCD) with incubation time. AWCD represents the mean color response for all 95 response wells. Plotted are the responses of the different treatment combinations.

tially important functional changes in response to elevated CO*. Others have attempted to detect shifts in the microbial community under elevated CO2. Zak et al. (1996) analysed microbial communities under elevated COz. They studied soil microbial responses in rhizosphere and non-rhizosphere soil under Populus grandidentata, using phospholipid fatty acid analysis (PLFA). With PLFA, the authors could not detect any significant CO2 effects on specific or nonspecific soil bacteria1 PLFA markers, and concluded that the microbial community composition did not change. It is possible that PLFA was not sensitive enough to detect changes at the level of phospholipid fatty acids, from which functional changes may be inferred. Biolog, however, directly measures patterns of oxidation and therefore may be a more suitable method. A direct comparison of the two methods is necessary to explore this possibility further. Although the Biolog analysis has been shown to produce consistent results in model bacterial communities (Haack et al., 1995) inferences about the actual microbial community in soil are problematic. This method can only capture the extractable, culturable bacteria that are able to use the sole carbon sources under the incubation conditions provided. Additionally, the 95 C sources are not selected to reflect rhizosphere substrates. Therefore, patterns that emerge from Biolog analysis should be interpreted with care and considered hypothesis-generating rather than hypothesis-testing (Bossio and Scow, 1995). Nevertheless, Biolog microplate analy-

Elevated CO? and soil microbial C utilization

sis was sensitive enough to detect changes in sole C utilization in response to an elevated CO2 treatment. This is important to note, since previous uses of the method included rather strong treatments (flooding, rice straw incorporation) or comparisons of microbial communities from different habitats (Garland and Mills, 1991. 1994; Zak er al.. 1994; Bossio and Scow, 1995). Eigenvalues for the principal component axes in PCA reflect the amount of variability in the data extracted by those axes. The eigenvalues reported here are similar to those found in previous Biolog studies. This shows that the C02-effect explained a significant proportion of the data. The separation of clusters of data points in the PCA is not as clear as in previous studies using Biolog, implying that the treatment is a somewhat more subtle one, compared to, for example. flooding. Given that CO2 was acting only indirectly on the soil microbial community, this is not very surprising

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In our experiment, we did not observe any increase in bacterial numbers with elevated C02, using a vital staining procedure. However, we noted an increase in AWCD, which is an estimate of the overall activity of the community cultured on the Biolog plates (Garland and Mills, 1991). This could mean that (a) total rhizodeposition did not increase or insufficiently so to induce cell proliferation, or that (b) the rhizosphere bacteria were limited by other resources. Most likely, they were limited by water availability (3.8% moisture content at final harvest). This could have important implications for semi-arid and arid ecosystems, where the soil microbial community may not be able to utilize fully the hypothesized increased supply of C. The fact that bacterial numbers did not respond to the N fertilization makes it appear unlikely that N was directly limiting the bacterial community in this experiment. However, N was limiting to plant growth. Therefore, N may have a long-term effect on the

Phosphorylated compounds

Alcohols

AmineS

Aromatic compounds

Amino acids

Carbohydrates

Ins

nsa

Amides

]nS *** ***

Carboxylic acids

H

72 h reading

H

96 h reading

0

120 h reading

ns

**

Esters

Ins

Polymers

i

0

I 1.5

I 2.0

1 2.5

, 3.0

Proportional change in oxidation in elevated CO2

Fig. 5. Proportional change in well color development on Biolog plates (oxidation) for different substrate groups when plants were exposed to elevated CO2. Different bar patterns denote different plate reading times. ***P< 0.001; **P < 0.01; *P < 0.05; ns. not significantly different from ambient CO2 (ANOVA).

Matthias C. Rillig et al.

1394

microbial community through its influence on plant growth and rhizodeposition. In conclusion, we propose that the measurement of merely soil microbial biomass in global change studies be supplemented by an effort to elucidate changes in microbial community structure and function. Acknowledgements-We thank Debbie Bossio, Jenn Macalady and Tessy Harizanos for expert technical assistance. M. C. R. acknowledges a doctoral fellowship from the Studienstiftung des deutschen Volkes (Bonn, Germany). This work was supported by a grant from the Program for Ecosystem Research, U.S. Department of Energy.

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