1 1 2 3 Lactate and Acrylate Metabolism by Megasphaera elsdenii Under Batch 4 and Steady State ...

30 Pages·2012·0.66 MB·English

AEM Accepts, published online ahead of print on 28 September 2012
Appl. Environ. Microbiol. doi:10.1128/AEM.02443-12
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
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4 Lactate and Acrylate Metabolism by Megasphaera elsdenii Under Batch
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5 and Steady State Conditions
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6 Rupal Prabhu1, Elliot Altman2, Mark A. Eiteman1, 3 n
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8 fr
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9 1) Dept. of Microbiology h
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10 University of Georgia, Athens, GA 30602 :/
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11 2) Dept. of Biology m
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12 Middle Tennessee State University m
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13 Murfreesboro, TN 37132 rg
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14 3) BioChemical Engineering, College of Engineering n

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15 University of Georgia, Athens, GA 30602 n
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18 Corresponding author: Mark A. Eiteman 9

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19 [email protected]
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20 Running Title: M. elsdenii lactate and acrylate metabolism s
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21 Abstract
22 The growth of Megasphaera elsdenii on lactate with acrylate and acrylate analogues was studied
23 under batch and steady-state conditions. Under batch conditions, lactate was converted to
24 acetate and propionate, and acrylate was converted into propionate. Acrylate analogues 2-methyl
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25 propenoate and 3-butenoate containing a terminal double bond were similarly converted into
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26 their respective saturated acids (isobutyrate and butyrate) while crotonate and lactate analogues n
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27 3-hydroxybutyrate and (R)-2-hydroxybutyrate were not metabolized. Under carbon-limited
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28 steady-state conditions, lactate was converted to acetate and butyrate with no propionate formed. fr
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29 As the acrylate concentration in the feed was increased, butyrate and hydrogen formation h
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30 decreased and propionate was increasingly generated, while the calculated ATP yield was :/
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31 unchanged. M. elsdenii metabolism differs substantially under batch and steady-state m
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32 conditions. The results support the conclusion that propionate is not formed during lactate- m
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33 limited steady-state growth because of the absence of this substrate to drive the formation of rg
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34 lactyl CoA via propionyl CoA transferase. Acrylate and acrylate analogues are reduced under n

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35 both batch and steady-state growth conditions after first being converted to thioesters via n
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36 propionyl CoA transferase. Our findings demonstrate the central role that CoA transferase y

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37 activity plays in the utilization of acids by M. elsdenii, and allows us to propose a modified ,
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38 acrylate pathway for M. elsdenii. 9

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40 Introduction
41 Megasphaera elsdenii is an ecologically important rumen bacterium which has recently
42 been sequenced (20) and which metabolizes DL-lactate principally to propionate and acetate (5,
43 7, 19). Lactate conversion to propionate occurs via the acrylate pathway with acrylyl-CoA
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44 serving as an intermediate (32), a pathway also used by several other organisms including
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45 Clostridium propionicum. The dead-end reduction of lactate to propionate allows the cell to n
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46 balance the anaerobic oxidation of lactate to acetate and carbon dioxide (16), steps which appear
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47 to be the primary means of ATP generation (26). M. elsdenii also produces butyrate, and several fr
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48 strains also accumulate longer chain fatty acids from the fermentation of lactate (9). The h
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49 generation of butyrate from lactate relies on the presence of acetate, and M. elsdenii also has the :/
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50 flexibility to generate hydrogen from reduced ferrodoxin as another means to balance redox (11). m
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51 While M. elsdenii continues to be of great interest as a member of the rumen microbial m
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52 community (21, 31), the organism and its enzymes also have potential biotechnological rg
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53 applications (28). n

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54 Key steps in the metabolic pathway of M. elsdenii to reduce lactate are mediated by n
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55 propionyl CoA transferase (24), lactyl CoA dehydratase (2,14,17) and acrylyl CoA reductase (3, y

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56 10). Propionyl CoA transferase (EC 2.8.3.1, systematic name acetyl CoA:propionate CoA- ,
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57 transferase) is typically implicated in the interconversion of propionate/propionyl CoA and DL- 9

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58 lactate/DL-lactyl CoA (26). However, this enzyme is a general CoA-transferase which can
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59 mediate other thioester exchanges including acetate/acetyl CoA and butyrate/butyryl CoA (24). s
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60 Lactyl CoA dehydratase (EC 4.2.1.54) has relatively low specific activity in dehydrating lactyl
61 CoA to acrylyl CoA (26), and can also mediate the reverse hydration (17). Acrylyl CoA appears
62 not to accumulate appreciably because of the abundance of acrylyl CoA reductase (EC 1.3.1.B1)
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63 in the soluble protein fraction and its very low K (10), enabling the propionyl CoA that is
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64 generated to achieve an equilibrium with propionate and other acid/thioester pairs via the
65 transferase. Although the enzyme propionyl CoA transferase from M. elsdenii may be involved
66 in various thioester/acid interconversions, the effect of other substrates on microbial physiology
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67 has not been established.
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68 Previous studies with pure cultures of M. elsdenii using lactate as a carbon source have n
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69 primarily investigated cells grown in batch culture. Under batch conditions, lactate is present in
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70 excess for essentially the duration of growth. Washed cell suspensions of M. elsdenii also fr
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71 ferment lactate and acrylate to yield acetate and propionate as products (18). M. elsdenii is h
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72 similarly able to grow on sugars like glucose and sucrose, although from these substrates :/
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73 butyrate is the principal end product, not propionate (13, 19). Although continuous cultures of m
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74 M. elsdenii have been completed (23), these studies focused on maintenance and energetics and m
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75 not on product formation. With Clostridium neopropionicum, which also uses the acrylate rg
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76 pathway, Seeliger et al. (27) reported no butyrate formation and observed no significant n

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77 difference in the proportion of propionate and acetate between continuous and batch processes n
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78 on lactate. Because of its ability to metabolize acrylate and the growing use of M. elsdenii y

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79 enzymes for biotechnological applications, the goals of this study were to investigate the ,
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80 metabolism of acrylate under both batch and steady-state continuous culture. 9

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81 Materials and Methods
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82 Strain and Media s
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83 M. elsdenii strain ATCC 17753 was used in this study. The medium used for serum
84 bottle/culture tube studies contained (per liter): 7.7 g D,L-lactate (86 mM), 3.0 g peptone, 4.0 g
85 yeast extract, 0.3 g cysteine·HCl, 0.1 g MgSO ·7H O, 0.018 g FeSO ·7H O, 5.0 ml of 1 mol l-1
4 2 4 2
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86 potassium phosphate pH 7.0, 2.5 ml saturated CaSO , and 0.1 mg resazurin. The gas atmosphere
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87 was 100% N . For batch and steady-state fermentations in the bioreactor, the medium contained
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88 (per liter): 3.15 g D,L-lactate (35 mM) and 0-2.5 g acrylate (0-35 mM), 0.6 g yeast extract, 0.5 g
89 cysteine·HCl, 40 ml mineral solution, and 1 ml vitamin solution. The mineral solution contained
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90 (per liter): 0.20 g CaCl , 0.20 g MgSO ·7H O, 1.00 g K HPO , 1.00 g KH PO , 10.0 g NaHCO
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91 and 2.00 g NaCl. The vitamin solution contained (per liter): 0.5 mg biotin, 20 mg pyridoxine, 20 n
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92 mg calcium pantothenate. The pH of the medium for bioreactor studies was adjusted to 6.5 using
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93 NaOH. fr
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94 Growth Conditions h
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95 M. elsdenii was routinely cultured in 150 ml serum bottles containing 100 ml of medium. :/
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96 To study the effect of acrylate on the growth rate, cultures were first grown without acrylate for m
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97 about 15 h (OD ~ 0.5) in serum bottles, then 0.5 ml of this culture was used to inoculate an m
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98 identical medium in a 10 ml tube which could be directly inserted into the spectrophotometer. rg
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99 When this second culture attained an OD of 0.1, acrylate (0 - 35 mM) was added anaerobically n

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100 with negligible volume change. Several chemical analogues of acrylate and lactate were n
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101 similarly examined by addition of 5 mM when the culture attained an OD of 0.1: 2-methyl y

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102 propenoate, 3-butenoate, crotonoate, 3-hydroxybutyrate and (R)-2-hydroxybutyrate. For the ,
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103 measurement of growth rate, samples were taken every 15 min for 2 h and also stored at -20°C 9

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104 for subsequent chromatographic analysis.
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105 Batch studies using 1.0 l medium volume were conducted in a 2.5 l bioreactor (Bioflo III s
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106 New Brunswick Scientific Co. Edison, NJ, USA). In this case, 50 ml of culture initially grown in
107 a serum bottle was transferred to 950 ml bioreactor medium. Nitrogen was sparged at 0.1 l/min
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108 throughout the process, and the agitation was 150 rpm. The temperature was maintained at 37°C,
109 and the pH was controlled at 6.5 with 15% HCl.
110 Continuous, steady-state fermentations of 1.0 l volume in a 2.5 l bioreactor operated at a
111 dilution rate of 0.125 h-1 as carbon-limited chemostats after initiation in the batch mode as
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112 described above. A steady state condition was assumed after four residence times at which time
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113 the CO concentration in the effluent gas remained unchanged. The temperature was maintained n
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114 at 37°C, the pH was controlled at 6.5 using 15% HCl.
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115 Analytical Methods r
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116 The optical density at 600 nm (OD) (UV-650 spectrophotometer, Beckman Instruments, h
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117 San Jose, CA, USA) was routinely used to monitor cell growth. Dry cell weight was measured :/
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118 by centrifuging/washing a 40-100 mL sample three times in deionized water and drying for 24 h m
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119 at 60°C. The concentration of carbon dioxide in the off-gas was measured using a gas analyzer m
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120 (Innova 1313 gas monitor, Lumasense Technologies, Ballerup, Denmark). Hydrogen was rg
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121 quantified in the effluent using gas chromatography (6850 Network GC system, Agilent n

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122 Technologies, USA) as previously described (25). Concentrations of soluble organic compounds n
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123 were determined by high performance liquid chromatography using a refractive index detector, y

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124 comparing unique retention times and integrated areas to pure standards for each analyte at five ,
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125 concentrations (0.5 – 10 mM) (6). 9

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126 Enzyme assays
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127 Propionyl-CoA transferase was measured at 25°C in 1.0 ml containing (final s
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128 concentrations) 100 mM potassium phosphate pH 7.0, 200 mM sodium acetate, 1.0 mM
129 oxaloacetate, 1.0 mM 5,3'- dithiobis(2-nitrobenzoate), 20 µg/ml citrate synthase and 0.1 mM
130 propionyl-CoA (Sigma-Aldrich Chemical Co., St. Louis, USA) (4). The reaction was initiated by
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131 addition of the cell extract, and the increase of absorbance was followed at 405 nm (Δε = 13.6
132 l/mmol·cm). Acetate kinase activity was measured colorimetrically at 540 nm by the reaction of
133 acetate phosphate with ferric iron (22). The components were 100 mM Tris·HCl buffer (pH 7.4),
134 10 mM ATP, 10 mM MgCl , 700 mM hydroxylamine and 800 mM potassium acetate. The
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135 reaction mixture was maintained at 37°C for 5 minutes, then cell extract added to initiate the
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136 reaction, and 10% trichloroacetic acid added to terminate the reaction. Butyrate kinase was n
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137 measured as described (8). For these enzymes one unit (U) of activity is the amount of enzyme
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138 which generates 1 µmol of product per minute. fr
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139 Results h
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140 Utilization of unsaturated short-chain acids :/
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141 M. elsdenii is a rumen bacterium capable of growing on DL-lactate which accumulates as m
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142 a result of a high fermentable sugar diet (5). M. elsdenii (12), as well as Clostridium m
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143 propionicum (15) and several other organisms, use the acrylyl CoA pathway and metabolize rg
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144 lactate and acrylate. We first compared M. elsdenii metabolism of lactate and acrylate under n

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145 batch conditions in serum bottles and also investigated whether this organism metabolized n
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146 analogues of acrylate and lactate. Specifically, we examined the unsaturated acids 2-methyl y

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147 propenoate (2-MP), 3-butenoate (3-BA), and trans-2-butenoate (crotonate) and the hydroxyl ,
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148 acids 3-hydroxybutyrate and (R)-2-hydroxybutyrate. Of all these organic acids, only lactate 9

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149 supported growth as the sole carbon source (data not shown). When growth was initiated on
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150 lactate and one of these compounds added, several of these organic acids were metabolized, and s
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151 Table 1 shows their mean product yields and relative substrate consumptions. M. elsdenii did
152 not utilize crotonate, 3-hydroxybutyrate or (R)-2-hydroxybutyrate added to a lactate
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153 fermentation, although none of these compounds affected continued growth on lactate (data not
154 shown).
155 When lactate was the sole carbon source in serum bottles, this carbon source partitioned
156 between acetate and propionate at a ratio of about 4:5. Assuming CO was generated by the
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157 formation of acetate and two moles of acetyl CoA were used to generate butyrate, the carbon
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158 recovery was 91%. When 3-carbon acrylate was added to the fermentation, the extent of acrylate n
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159 consumption was 74% on a molar basis of the extent of lactate consumption, and the molar yield
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160 of acetate and propionate was again in the ratio of about 4:5 with 89% carbon recovery. The fr
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161 branched acid 2-MP was consumed to an extent of 55% of the lactate consumed, and the h
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162 branched saturated acid isobutyrate was observed as a product (87% recovery). About 1.78 :/
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163 times as much 3-BA was consumed on a molar basis as lactate after this unsaturated acid had m
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164 been added to the growing culture, and butyrate was observed to increase proportionately (93% m
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165 recovery, assuming newly observed butyrate is derived from 3-BA). rg
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166 Batch growth on lactate and acrylate n

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167 In preparation for batch studies using both lactate and acrylate, we next determined the n
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168 growth rate of M. elsdenii in serum bottles on lactate in the presence of acrylate. We found the y

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169 maximum specific growth rate in the absence of acrylate to average 0.62 h-1. The μMAX declined , 2
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170 only slightly with increasing acrylate concentrations to 10 mM (Figure 1). Above 10 mM 9

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171 acrylate μ declined more substantially, resulting in a growth rate of less than 0.15 h-1 at 30
MAX g
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172 mM acrylate. No growth was observed in the presence of 35 mM or greater acrylate. s
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173 The growth and product distribution of M. elsdenii on lactate with and without added
174 acrylate was compared in anaerobic batch cultures in a controlled bioreactor (about 55 mM total
175 acid concentration). In the absence of acrylate, the final molar proportion of
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176 acetate:propionate:butyrate generated was 10:8.0:4.7 (Figure 2). Similar to previous
177 observations, butyrate accumulated at a greater rate late in the fermentation. Considering that
178 CO was generated by the formation of acetyl CoA (and acetate) and two moles of acetyl CoA
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179 were used to generate butyrate, the carbon recovery for the process was 93%, with 66% of the
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180 lactate flux partitioning to acetate/butyrate and 27% of the lactate flux partitioning to propionate.
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181 Assuming one mole of ATP was generated with each mole of butyrate and acetate formed, the n
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182 cell yield on ATP (Y ) was measured to be 12.5 g cells/mol ATP.
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183 In the presence of 20 mM acrylate in the bioreactor, the batch growth of M. elsdenii was fr
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184 slowed as expected due to reduced tolerance to acrylate (Figure 3). Nevertheless, the h
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185 consumption of acrylate was initially much faster than the rate of lactate consumption. For :/
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186 example, during the first 12.5 h of growth, 4.9 mM lactate and 17.2 mM acrylate had been m
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187 consumed, while 10.0 mM acetate, 13.2 mM propionate and 0.4 mM butyrate had been m
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188 generated. The carbon recovery for this portion of the process was 109% with 49% of the rg
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189 lactate/acrylate partitioning to acetate/butyrate and 60% partitioning to propionate. The Y n
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190 during this portion was 12.5 g cells/mol ATP. Between 12.5 h and 23.5 h, the remaining n
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191 substrates were consumed (27.7 mM lactate and 2.6 mM acrylate), while 6.6 mM acetate, 4.1 y

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192 mM propionate, 4.3 mM butyrate and only an additional 0.05 g/l cells were generated. ,
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193 Steady-state growth on lactate and acrylate 9

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194 We next performed several carbon-limited chemostats at a growth rate of 0.125 h-1 to y
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195 compare steady-state metabolism in the presence of increasing acrylate concentrations in the feed s
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196 (0, 10 and 20 mM). Because M. elsdenii showed a maximum growth rate far above a dilution
197 rate of 0.125 h-1 with 20 mM acrylate (Figure 1) and because acrylate was readily metabolized in
198 batch culture (Figure 3), we anticipated that acrylate would be depleted under carbon-limited
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199 steady-state conditions, which was indeed the case (steady-state concentrations about 1 mM). In
200 addition to measuring organic acid products, we measured hydrogen and CO in the effluent gas.
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201 We were unable to attain a steady-state when the acrylate concentration was above 20 mM in the
202 presence of 35 mM lactate.
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203 With lactate as the sole carbon source (i.e., no acrylate), essentially no propionate formed
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204 in duplicate experiments under steady-state conditions. Instead, butyrate was the primary acid n
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205 product. We observed a mean specific lactate consumption rate (q ) of 39.2 mmol/g·h with
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206 products formed at a rate of 4.6 mmol acetate/g·h, <0.5 mmol propionate/g·h, 10.4 mmol fr
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207 butyrate/g·h, 45.6 mmol CO2/g·h, 31.1 mmol H2/g·h (88% carbon recovery). Assuming again h
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208 that acetate and butyrate formation generated ATP, the YATP was 8.3 g cells/mol ATP. ://
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209 As expected, both lactate and acrylate were consumed in the steady-state processes m
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210 containing both substrates, and increasing the proportion of acrylate from 0 mM to 20 mM in the m
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211 feed therefore increased the fraction of acrylate consumed relative to the total organic acids . In rg
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212 order to quantify the effect of an increasing acrylate proportion in the mixture, we defined the n

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213 molar acrylate fraction (i.e., subscript ACR) consumed ξ = q /(q +q ) where q is the n
ACR ACR ACR LAC u
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214 specific rate of substrate consumption and ξ + ξ = 1. Then, the fraction ξ of that total y
ACR LAC I
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215 molar substrate flux which partitioned toward a particular product I was calculated. For ,
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216 example, considering a mole of acetate is generated from one mole of lactate or acrylate, the 9

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217 fractional flux of lactate or acrylate to form acetate is ξACE = qACE/(qACR+qLAC). Similarly, 1 g
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218 mole of butyrate requires two moles of either substrate so that ξBUT = 2×qBUT/(qACR+qLAC). s
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219 Increasing the proportion of acrylate consumed under steady-state conditions from an
220 acrylate-lactate mixture (ξ ) increased propionate and acetate formation (ξ , ξ ) and
ACR LAC ACE
221 decreased butyrate formation (ξ , Figure 4). For example and as noted above, in the absence
BUT
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