Improvement of Lactobacillus plantarum Aerobic Growth as Directed by Comprehensive Transcriptome Analysis

An aerobic Lactobacillus plantarum culture displayed growthstagnation during early growth. Transcriptome analysis revealedthat resumption of growth after stagnation correlated with activationof CO₂-producing pathways, suggesting that a limiting CO₂ concentrationinduced the stagnation. Analogously, increasing the CO₂ gaspartial pressure during aerobic fermentation prevented the temporalgrowth stagnation.

INTRODUCTION

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INTRODUCTION

Lactobacillus plantarum is a facultative heterofermentativelactic acid bacterium used worldwide in production of fermentedfood and feed products, and its natural habitats are anaerobicor microaerobic. Nevertheless, the responses of L. plantarumto aerobic growth conditions and the corresponding oxidativestresses are relevant for a variety of industrial processingconditions (10). Moreover, a potential ability to respire andincrease the biomass yield during aerobic growth has been demonstratedfor several lactic acid bacteria, including Lactococcus lactis(2, 5). L. plantarum is able to utilize oxygen under certaincircumstances, including glucose limitation conditions (6),and the absence of the ubiquitous defensive reaction catalyzedby superoxide dismutase in L. plantarum was found to be compensatedfor by the capacity of this bacterium to accumulate very highconcentrations of intracellular Mn(II) ions (up to 35 mM), whichact as a scavenger system for superoxide (1, 8).

In this work an aerobically grown culture showed consistenttemporary growth stagnation during the early logarithmic phase.Transcriptome analyses revealed genes that were differentiallyexpressed before and after the growth stagnation, and comprehensiveanalysis of these differentially expressed genes revealed thatresumption of growth after the observed growth stagnation correspondedwith the activation of a range of CO₂-producing metabolic pathways,suggesting that the growth stagnation was due to CO₂ limitation.Correspondingly, it could be shown that modifying the gas supplementationregimen by increasing the CO₂ partial pressure relieved growthstagnation, confirming the CO₂ limitation hypothesis.

Growth of L. plantarum under aerobic conditions.

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Growth of L. plantarum...

L. plantarum strain WCFS1 (9) was grown aerobically and anaerobicallyat 37°C in MRS medium (3), and growth was monitored for12 h (Fig. 1). L. plantarum displayed two phases of logarithmicgrowth (Fig. 1), which is in agreement with previous observations(4). The final cell density was higher in the aerobic culture(optical density at 600 nm [OD₆₀₀], 5.5) than in the anaerobicculture (OD₆₀₀, 4.5). Interestingly, temporary growth stagnationoccurred during aerobic fermentation after approximately 2 h,a feature not observed in the anaerobic culture (Fig. 1). Followingthis stagnation, growth resumed, and the maximum growth ratewas comparable to that observed in anaerobic cultures (Fig.1).

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FIG. 1. Growth characteristics of L. plantarum in batch fermentations under anaerobic conditions (N₂ headspace) and under aerobic conditions (air flushed at a rate of 1 volume/min). Symbols: ${triangleup}$ , optical density of the anaerobic culture; ${blacktriangleup}$ , acidification of the anaerobic culture; •, optical density of the aerobic culture; ${circ}$ , acidification of the aerobic culture. The sampling points for transcriptome analyses are indicated: P1 (OD₆₀₀, $~$ 0.4) and P2 (OD₆₀₀, $~$ 1.0).

The high growth rates and rapid acidification of the mediumin the early growth phase, apparently irrespective of the presenceof oxygen, confirmed previously described results (6, 7). Moreover,the significantly altered physiology of aerobically grown L.plantarum cells compared to anaerobically grown cells as a consequenceof the presence of oxygen was illustrated by the higher celldensity reached in the aerobic culture and the fermentationpattern slightly shifted toward acetate production (6 mM acetatewas produced aerobically, and 2 mM acetate was produced anaerobically).However, the growth stagnation observed under aerobic conditionscould not be readily explained, and we decided to study thisphenomenon further.

Changes during the early growth phase of an aerobic culture.

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Changes during the early...

As described above, L. plantarum displayed temporary growthstagnation in the early aerobic growth phase (Fig. 1). Sincethe precultures used to inoculate the aerobic fermentation cultureswere already adapted to aerobic growth, it seems likely thata limitation of the growth medium and not the presence of oxygenper se caused the growth stagnation. Notably, growth stagnationalways appeared to occur approximately 100 min after the initiationof fermentation, independent of the inoculum size (Fig. 2).Nevertheless, inoculation at a higher density resulted in shorterstagnation periods, and the duration of the growth stagnationcorrelated inversely with the inoculum size (Fig. 2). Thesecharacteristics suggest that the growth stagnation observedwas due to limitation of a specific medium component that wasflushed out by aeration during the initial phase of fermentation.

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FIG. 2. Temporary stagnation of growth during aerobic growth and dependence of the duration of the stagnation on inoculum size. The results for three cultures, inoculated at OD₆₀₀ of 0.2, 0.1, and 0.02, are shown. Clearly, the duration of the stagnation increased with decreasing initial optical density (arrows). Representative graphs for experiments performed in triplicate are shown.

Full-genome transcriptional analyses of aerobic and anaerobic cultures.

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Full-genome transcriptional...

To elucidate the processes that caused the growth stagnationin the aerobic culture, genome-wide transcription analyses wereperformed using amplicon-based DNA microarrays (GEO accessionno. GPL6368 [see the supplemental material]). Cells used fortranscriptome analyses were harvested before and after the growthstagnation using a quenching method to minimize changes in thetranscriptome during harvesting (11). RNA was subsequently isolated,transcriptome experiments were performed, and the results wereanalyzed as described in the supplemental material.

The transcriptome profiles (GEO accession no. GSE10194) obtainedbefore growth stagnation (P1, corresponding to an OD₆₀₀ of $~$ 0.4)and after resumption of growth following the stagnation period(P2, corresponding to an OD₆₀₀ of $~$ 1.0) (Fig. 1) were projectedon a metabolic map using a metabolic model for L. plantarumWCFS1 (13). This projection revealed several pyruvate-associatedmetabolic pathways that are expressed at a higher level in P2than in P1, including pyruvate dehydrogenase and malate dehydrogenasepathways (Fig. 3A; see the supplemental material). Interestingly,these pathways include a reaction that produces CO₂, while athird CO₂-producing pathway that involves pyruvate oxidase displayeda similar trend of expression modulation, albeit with lowersignificance (P = 0.11) (Fig. 3B; see the supplemental material),suggesting that CO₂ production is enhanced in P2 compared toP1. CO₂ is essential in purine biosynthesis during productionof the intermediate 5-amino-1-ribosylimidazole 5-phosphate.Moreover, the reaction catalyzed by carbamoyl-phosphate synthase,which is involved in pyrimidine and arginine synthesis, requiresthe dissolved form of carbon dioxide (HCO₃^–) (Fig. 3B).The data suggest that limiting CO₂ concentrations lead to growthstagnation due to their impact on the rates of biosynthesisof nucleotides. Moreover, the data suggest that CO₂ is not limitingduring the early growth phase (P1), since high growth ratesare sustained without induction of the CO₂-producing pathways.Time-dependent CO₂ limitation could occur by stripping of theCO₂ in the medium as a consequence of the high gas flush rateimposed by aerobic conditions. The induction of CO₂-producingpathways appears to indicate that the bacterium initiates endogenousCO₂ production to compensate for the limiting environmentalavailability of this compound.

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FIG. 3. (A) Transcriptome data for aerobic P1 versus P2 projected on a metabolic map of pyruvate metabolism of L. plantarum. The filled triangles indicate reactions corresponding to upregulated genes in P2, the gray boxes indicate no significant regulation, and a multiplication sign indicates that no data are available. All reactions were significantly regulated (P < 0.05), except for the pox gene reaction (P = 0.11). Abbreviations: 2PG, 2-phosphoglycerate; Pep, phosphoenolpyruvate; Pyr, pyruvate; L-Lac, L-lactate; D-Lac, D-lactate; Mal, malate; Fum, fumarate; OAA, oxaloacetate; Cit, citrate; EtOH, ethanol; AcAld, acetaldehyde; Ac, acetate; Ac-P, acetyl phosphate; AcCoA, acetyl coenzyme A; For, formate. (B) Carbon dioxide-consuming and -producing reactions in L. plantarum. Enzymes are differentially expressed at time points P1 and P2 in aerobic cultures. The open arrows indicate reactions corresponding to upregulated genes in P2, the gray arrows indicate no significant regulation. All reactions were significantly regulated (P < 0.05), except for the pox gene reaction (P = 0.11). Abbreviations: MAE, malic enzyme; PDH, pyruvate dehydrogenase; Pox, pyruvate oxidase; PurK, phosphoribosylamino-imidazole carboxylase; Cah, carbonate anhydrase; PyrAA, carbamoyl-phosphate synthase; AccC, acetyl-coenzyme A carboxylase; PycA, pyruvate carboxylase.

Improved fermentation of aerobic L. plantarum.

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Improved fermentation of aerobic...

To investigate whether CO₂ limitation causes the growth stagnationobserved during the early logarithmic growth phase, L. plantarumWCFS1 was cultured aerobically and the culture was flushed withair or with air enriched with 1% CO₂. To emphasize the growthstagnation, the culture was inoculated so that the initial OD₆₀₀was approximately 0.1 (Fig. 4; also see above). In the cultureflushed with normal air, growth stagnation occurred as usualafter approximately 100 min (Fig. 4), and due to the lower initialOD₆₀₀ it lasted for at least 2.5 h. In contrast, the cultureflushed with 1% CO₂-enriched air displayed no growth stagnation(Fig. 4), confirming that CO₂ limitation hampers continuousgrowth of aerobically cultured L. plantarum, which can readilybe compensated for by supplementation with additional CO₂.

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FIG. 4. Elimination of the temporal growth stagnation by increased CO₂ level. The culture flushed with air (1 volume/min) showed the typical temporary growth stagnation ( ${blacksquare}$ ), whereas growth stagnation did not occur in the culture flushed with air mixed with 1% CO₂ ( ${square}$ ).

The metabolic model for L. plantarum WCFS1 predicts that bicarbonateis used for the production of purines and pyrimidines (12).The solubility of carbonate at 37°C and at pH <6.5 islow, and intense flushing of media with air leads to a decreasein the carbonate concentration, ultimately causing growth stagnationin cultures flushed with air (Fig. 1) or nitrogen (data notshown). The dependence of growth on the concentration of CO₂or carbonate was confirmed by the resumption of growth whenthe culture was flushed with air containing 1% additional CO₂.

Our analyses indicate the power of postgenomic approaches fordiscovering bacterial fermentation- or growth-limiting factors.They provide a first step for directed adjustment of fermentationconditions to improve bacterial performance.

ACKNOWLEDGMENTS

This work was supported by grant IGE1018 from the Dutch IOP-GenomicsProgram.

FOOTNOTES

* Corresponding author. Mailing address: NIZO food research, P.O. Box 20, 6710 BA Ede, The Netherlands. Phone: 31-(0)318-659629. Fax: 31-(0)318-650400. E-mail: Michiel.kleerebezem{at}nizo.nl

Published ahead of print on 6 June 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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