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Enhanced methane production from food waste using cysteine to increase biotransformation of L-monosaccharide, VFA and bio-hydrogen Hui Liu, and Yinguang Chen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05355 • Publication Date (Web): 21 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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Environmental Science & Technology

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Enhanced methane production from food waste using cysteine to increase

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biotransformation of L-monosaccharide, VFA and bio-hydrogen

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Hui Liu, Yinguang Chen*

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( State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China)

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* Corresponding author

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E-mail: [email protected]

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Tel.: 86-21-65981263

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Fax: 86-21-65986313

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ACS Paragon Plus Environment


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Abstract: The enhancement of two-stage anaerobic digestion of polysaccharide enriched food waste by

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the addition of cysteine, an oxygen scavenger, electron mediator and nitrogen source, to the acidification

13

stage was reported. It was found that in the acidification stage the accumulation of volatile fatty acids

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(VFA), which mainly consisted of acetate, butyrate and propionate, was increased by 49.3% at cysteine

15

dosage 50 mg/L. Although partial cysteine was biodegraded in the acidification stage, the VFA derived from

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cysteine was negligible. In the methanogenesis stage, the biotransformations of both VFA and bio-hydrogen

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to methane were enhanced and the methane yield was improved by 43.9%. The mechanisms study showed

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that both D-glucose and L-glucose (the model monosaccharides) were detectable in the hydrolysis product,

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and the addition of cysteine remarkably increased the acidification of L-glucose, especially acetic acid and

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hydrogen generation due to key enzymes involved in L-glucose metabolism being enhanced. Cysteine also

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improved the activity of homoacetogens by 34.8% and hydrogenotrophic methanogens by 54%, which

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might be due to the electron transfer process being accelerated. This study provided an alternative method to

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improve anaerobic digestion performance and energy recovery from food waste.

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Introduction

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Anaerobic digestion is considered as a preferable method for reutilizing organic matters (mainly

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polysaccharide, protein and lipids) in wastes since it can produce energy (i.e., methane) with simultaneous

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reduction of wastes.1 Usually, three steps are involved in anaerobic digestion, i.e., hydrolysis, acidification,

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and methanogenesis. Firstly, organic macromolecule (polysaccharide, protein and lipids) in organic wastes

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are hydrolyzed to monosaccharide, amino acid and free fatty acids by anaerobic microbes. Then, the

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hydrolyzed products (such as glucose) are bio-converted to acetic acid and other volatile fatty acids (VFA,

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such as propionic and butyric acids) by acid-producing bacteria. Finally, methanogens use the generated

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acetic acid to produce methane and carbon dioxide under favorable anaerobic conditions. As hydrolysis is

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believed the rate-limiting step of anaerobic digestion, previous studies usually focus on the use of various

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pretreatment methods, such as alkaline pH and/or thermophilic temperature, to increase the hydrolysis

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efficiency of wastes for improving methane yield.2-5

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Monosaccharide (such as glucose) is the main hydrolysis product when polysaccharide-enriched

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organic wastes are digested.

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(dextorotatory, right-handed) and L (levorotatory, left-handed)), most publications only discuss or assay the

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monosaccharide with D-configuration, especially D-glucose, when hydrolysis step is studied, which might

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be due to the natural configuration of glucose being D-glucose.6,7 In fact, L-monosaccharide has also been

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reported to be generated during hydrolysis.

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hydrolysate of corn stalk.8 Also, L-monosaccharides, such as L-glucose, L-frutose and L-xylulose, have been

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used widely as low calorie sweeteners, bulking agents, and inhibitors of bacterial growth and glucosidases,9-

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12

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biodegradation behavior of L-monosaccharide.

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configuration of monosaccharide and the effect of monosaccharide’s configuration on acidification,

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especially on the amount and composition of VFA, have rarely been investigated.

Although monosaccharide has two types of configuration (i.e., D

For example, L-monosaccharide was observed in the

and they may release into the environment. Therefore, it is of vital importance to explore the anaerobic Until now, however, the influence of hydrolysis on

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Acetic acid and hydrogen are also the important intermediate products affecting anaerobic digestion.

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They are the direct substrates for methane production. Usually, acetic acid is derived from the acidification

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of monosaccharide. Nevertheless, it can also be formed from biohydrogen and carbon dioxide, which are

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generated in acidification stage by homoacetogens via Wood-Ljungdahl pathway according to equation 1.13-

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If the biotransformation of hydrogen and carbon dioxide to acetic acid in the acidification step could be 3



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increased, more acetic acid would be generated, which will provide more substrate for acetotrophic

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methanogens to produce more methane in the methanogenesis step. It was also reported that in the

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methanogenesis step other VFA (such as propionic and butyric acids) can be biodegraded to acetic acid with

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the generation of hydrogen (equations 2 and 3), and the generated hydrogen can be taken up together with

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CO2, coming from the activity of acetotrophic methanogens, by hydrogenotrophic methanogens to produce

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methane (equation 4).16-18 Clearly, improving the activities of microbes responsible for bioconversion of

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other VFA to acetic acid and hydrogen/carbon dioxide to methane will also be in favor of the increase of

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methane yield. Although several environmental and operational factors, such as trace element, humic acids,

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polycyclic aromatic hydrocarbon and pH value, have been reported to influence the microbial activity of

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anaerobic digestion, the strategy for simultaneously increasing the microbial activities of homoacetogens and

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acetotrophic and hydrogenotrophic methanogens has never been reported.19-24

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4H 2 + 2CO2 → CH 3 COOH + 2 H 2 O

(1)

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CH 3 CH 2 COOH + 2 H 2 O → CH 3 COOH + CO 2 + 3H 2

(2)

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CH 3 CH 2 CH 2 COOH + 2 H 2 O → 2CH 3 COOH + 2 H 2

(3)

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4 H 2 + CO 2 → CH 4 + 2 H 2 O

(4)

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Cysteine has been reported to have the properties of stabilizing oxidation-reduction potential, affecting

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catalytic activity, and making posttranslational modifications of some proteins.25 For example, it can lower

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the redox potential,26 scavenge oxgen,27 stimulate the reduction of iron (III) oxides by Geobacter

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sulfurreducens,28 affect biohydrogen production,29 mediate electron transfer between different guilds,28,30

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and act as nitrogen source supplier.31 Until now, however, the influence of cysteine on the digestion of food

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waste, which mainly comes from restaurants, hotels, canteens, and companies with a total amount of 1.6

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gigatonnes annually,32 has seldom been studied. It was observed in our study that the presence of certain

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amount of cysteine in the anaerobic digestion system could remarkably enhance methane production from

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food waste. The purpose of this study was therefore to report a new strategy for increasing methane

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production from food waste via the use of cysteine to improve the bioconversion of monosaccharide with L-

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configuration and the biotransformation of VFA and bio-hydrogen. As two-stage anaerobic digestion (i.e.,

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firstly fermenting organic wastes to generate VFA and then the VFA-enriched mixture being used as the

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substrate for methane production) usually can produce more methane than single-stage,5,33,34 it was applied 4


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to anaerobically digest food waste in this study. Firstly, the performance of anaerobic digestion of food

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waste for methane production enhanced by cysteine was reported. Then, the influences of cysteine on the

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hydrolysis of main organic compound of food waste, the bio-transformations of monosaccharide with

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different configuration, VFA, and hydrogen in acidification and methanogenesis stages, and the activities of

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key microbes and enzymes were investigated to explore the possible reasons for cysteine improving methane

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yield.

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Materials and methods

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Analytic methods. TS (total solid) and VS (volatile solid) were analyzed by standard methods.35 The VFA

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composition was analyzed by gas chromatography (Agilent 7820N) with a flame ionization detector and

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DB-WAXETR column (30 m×1.0 µm×0.53 mm). The sum of measured acetic, propionic, n-butyric, iso-

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butyric, n-valeric, and iso-valeric acids was recorded as the concentration of total VFA (mg COD/L) with

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proper COD conversion (i.e. 1 g acetic acid=1.07 g-COD, 1 g propionic acid=1.51 g-COD, 1 g butyric

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acid=1.82 g-COD, and 1 g valeric acid=2.04 g-COD).36

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hydrogen, were determined via a gas chromatograph (Agilent 6890N) equipped with a thermal conductivity

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detector using nitrogen as a carrier gas.15

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pyruvate-ferredoxin oxidoreductase, formate dehydrogenase, formyltetrahydrofolate synthetase) were

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assayed as described in our previous publications.15,37 For their determinations, 25 mL of the mixture was

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taken out of the reactors and then washed and resuspended in 10 mL of 100 mM sodium phosphate buffer

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(pH 7.4). The suspension was sonicated at 20 kHz and 4°C for 30 min to break down the cells of bacteria

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and then centrifuged at 10000 rpm and 4°C for 30 min to remove the waste debris. The extracts were kept

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cold on ice before enzyme activity was assayed. The content of ATP and the L-glucose dehydrogenase

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(LGDH) activity were measured according to the references.38,39 Other analytical methods are detailed in

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Supporting Information.

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Statistical analysis. All assays were conducted in triplicate and the results were expressed as mean ±

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standard deviation. An analysis of variance (ANOVA) was used to test the significance of results, and p
0.05), Figure 1B did not show a large difference

200

between VFA composition.

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The pH value and reaction time were reported to affect not only the biodegradation of refractory

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organic compounds which are difficult to be biodegraded, but the generation of VFA during acidification.37,46

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In the presence of cysteine, however, the optimal conditions for VFA generation from food waste have never

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been studied. As seen in Figure 1C the VFA concentrations at any pH value investigated were increased

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significantly with time (p0.05), suggesting that the suitable pH for food waste acidification was pH 6 and the

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acidification time was 8 d. Under the optimal conditions (i.e., cysteine dosage 50 mg/L, pH 6 and time 8d),

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the VFA concentration reached 18071 mg COD/L, and the top three compositions of VFA were acetic (8738

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mg COD/L), butyric (7784 mg COD/L), and propionic (1198 mg COD/L) acids, which accounted for 48.4%,

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43.1% and 6.6% of the total VFA, respectively.

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concentration of VFA under conditions of pH 6 and time 8 d was only 12104 mg COD/L, which indicated

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that the addition of cysteine caused a 49.3% enhancement of VFA accumulation in the acidification stage.

Without the supplement of cysteine, however, the

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The above acidified food waste (AFW) and cysteine enhanced acidified food waste (CE-AFW) were

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then used respectively as substrate to produce methane in two long-term operated methanogenesis reactors.

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Figure 2A shows the data of methane production during 60 day’s operation after the yield of methane

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reached relatively stable. The average yield of methane was 328 mL/g VS in the control reactor, and it was

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increased to 472 mL/g VS in the cysteine-enhanced acidified food waste (CE-AFW) methanogenesis reactor.

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The improvement of methane yield was 43.9%. Further analysis of COD balance was conducted (Table S2,

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Supporting Information), and it can be indicated that in the acidification stage about 35.9% of the influent

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(food waste) total COD was converted to VFA in the control reactor, while 53.7% of the influent total COD 9



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was converted to VFA in the cysteine addition reactor. In the methanogenesis stage, 43.1% (control) and

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62.6% (cysteine) of the influent (i.e., acidification liquid) total COD were converted to methane,

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respectively. Since the VFA production from food waste in acidification stage and VFA conversion to

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methane in methanogenesis step were both enhanced by cysteine addition, significant enhancement of

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methane yield was therefore observed.

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The methane production observed in this study was greater than that reported in the literature when

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food waste was digested in two-stage at mesophilic temperature (Table S3, Supporting Information).47-52

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Further measurement of ATP, which had been used to assess the general physiological activity of anaerobic

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cells as the increase of methane production was observed to be in correspondence with that of ATP,38,53

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showed that the average ATP concentration was 1.45 times higher (23.3 µg/L) in the CE-ATW reactor than

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that of the control (16.1 µg/L). As the trends between Fig 2A and Fig 2B were very similar over time, it

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seems that the use of cysteine-enhanced acidified food waste as the substrate for methane production

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improved the activity of methanogens. Cysteine can be served as a nitrogen source when the content of

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nitrogen is not enough for microbial activity. Usually the required nitrogen/carbon ratio (TN/BOD) for

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microbes is 5/(100-200), i.e., 1/20-40. In this study the ratios of TN/BOD in the food waste fermentation

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tests was 1/9.34, which were much greater than 1/(20-40). Thus, the production of methane enhanced by

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cysteine was not due to its service as nitrogen source. In the coming text the possible reasons for cysteine

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improving two-stage anaerobic digestion were explored. 30

B

AFW (Control)

CE-AFW

CE-AFW

25

500

ATP (ug/L)

Methane (ml/gVS)

A

AFW (Control)

600

400 300

20

15 200 100

10

0

10

20

30 40 Time (d)

50

60

0

10

20

30 40 Time (d)

50

60

240 241

Figure 2. Performance of methane production (A) and general physiological activity of anaerobic cells

242

measured by ATP (B) in the long-term methanogenesis experiments. Error bars represent standard deviations 10


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of triplicate tests.

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Effects of cysteine on hydrolysis of main organic compound of food waste. Polysaccharide is the top one

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organic compound in food waste, which can be hydrolyzed to monosaccharide in the hydrolysis stage.

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When glucose was considered as the model monosaccharide, it was observed in our study that not only D-

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glucose, the well know natural configuration of glucose, but also L-glucose was generated in the food waste

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during hydrolysis tests. The final D-glucose was respectively 943±24 and 996±32 mg COD/L in the absence

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(control) and presence of cysteine, and the corresponding L-glucose was 311±24 and 335±23 mg COD/L.

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The statistic analysis indicated that the addition of cysteine did not significantly affect the concentration of

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both D-glucose and L-glucose (p>0.05), indicating that the reason for cysteine improving VFA generation

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was not due to its influences on the hydrolysis of main organic compound of food waste.

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Protein is the second organic compound in food waste, but its content was much lower than

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polysaccharide (11.73 versus 81.96 g/L). In the hydrolysis stage protein is bio-converted to amino acids.

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The final total amino acids concentration in the hydrolysis liquid was 31.4 mg COD/L in the control test, and

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it was 58.9 mg COD/L after the addition of cysteine. Compared with D-glucose and L-glucose, the

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concentration of amino acids was much lower. Thus, this paper focused on the bio-transformation of

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polysaccharide and the influence of cysteine addition on protein hydrolysis was not considered.

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Effects of cysteine on bio-transformations of hydrolyzed product and hydrogen in acidification stage.

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The tests of cysteine affecting acidification of D-glucose showed that no matter whether cysteine was added

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or not, all D-glucose was completely consumed (Table 1). The measurement of acidification products

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revealed that acetic, propionic and butyric acids were detectable in the D-glucose acidification test, and

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acetic and butyric acids were the top two VFA. The total VFA concentration was 812.4 and 827.8 mg

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COD/L in the control and cysteine added tests, respectively. It can be calculated from Table 1 that acetic and

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propionic acids accounted respectively for 67.3% and 21.6% of total VFA in the control test, and 68.3% and

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20.2% in the cysteine one. It seems that cysteine had no obvious effect on acidification of D-glucose, which

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might be due to the fact that D-glucose is an easily biodegradable substrate. Nevertheless, in the L-glucose

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acidification test, the acidification efficiency of L-glucose was only 19.2% in the absence of cysteine, i.e., L-

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glucose was slightly decreased from 323 to 261 mg COD/L (Table 1). With the addition of cysteine,

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however, the final L-glucose concentration was declined to 12 mg COD/L, and the acidification efficiency

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reached 96.3%. By analyzing the cysteine content after acidification it was observed that there was still 20.2 11



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mg/L of cysteine, indicating that the maximal biodegradation of cysteine in the current acidification system

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was 29.8 mg/L. Even when all biodegraded cysteine was converted to acetic acid, the theoretic yield of

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acetic acid was only 15.8 mg COD/L, which was negligible. The VFA concentrations were respectively 51.3

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and 280.6 mg COD/L in the absence and presence of cysteine tests. From Table 1 it can also be seen that

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there were only acetic and propionic acids in the acidification liquid of L-glucose. The percentage of acetic

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and propionic acids was respectively 51.8% and 48.2% in the control test, but it was 60.1% and 39.9% in the

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cysteine addition test. The addition of cysteine to L-glucose acidification reactor increased the ratio of

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acetic/propionate from 1.08 to 1.51. Apparently, the acidification product of L-glucose was different with

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that of D-glucose. Also, it can be concluded that cysteine enhanced not only the acidification of L-glucose,

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but the percentage of acetic acid.

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Table 1.

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acidification tests. a

The influences of cysteine on the concentrations of D-glucose, L-glucose and VFA in the

Concentration of substrate Concentration of VFA in the final acidification liquid (D-glucose or L-glucose) Initial

284 285 286 287 288

b

Final

Acetic

Propionic

Butyric

c

Total VFA

D-glucose

Control

970±12

0

546.8±38.8

90.0±9.2

175.6±13.2

812.4±42.7

reactor

Cysteine

970±12

0

565.5±34.7

94.8±8.3

167.5±10.8

827.8±50.4

L-glucose

Control

323±9

261±11

26.6±3.5

24.7±3.5

0

51.3±6.3

reactor

Cysteine

323±9

12±2

168.6± 13.4

112.0± 10.3

0

280.6±20.9

a

The acidification time for L-glucose was 4 d, but it was 2 d for D-glucose as all D-glucose was consumed on day 2 in

acidification tests. The unit is mg COD/L. Apart from acetic, propionic, and butyric acids, there was no other VFA detectable in the final acidification liquid.

b

The initial concentrations of D-glucose and L-glucose in the acidification tests were decided

according to the average data of their final concentrations in the hydrolysis tests.

c

The sum of n-butyric and iso-butyric acids.

The metabolism of anaerobic biotransformation of L-glucose to acetic and propionic acids has never Shimizu et al.39 studied the anaerobic degradation of L-glucose by

289

been reported in the literature.

290

Paracoccus species to pyruvic acid and proposed that L-glucose was firstly bio-transformed to L-glucono-

291

1,5-lactone by LGDH (L-glucose dehydrogenase), which was then to L-gluconoate, L-5-ketogluconoate, D-

292

idonate, D-2-keto-3-deoxygalactonate (KDGal), D-2-keto-3-deoxy-6-phosphogalactonate (KDPGal), and

293

finally pyruvic acid.

294

generated in the acidification process in the presence of cysteine, and the key enzyme (L-glucose

295

dehydrogenase) was enhanced (Figure 3A). It seems that the metabolic pathway for the bio-conversion of L-

In the current study the chemical analysis indicated that there was pyruvic acid

12


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glucose to pyruvic acid reported in the literature was also present in our acidification system. On the other

297

hand, there were large amounts of acetic and propionic acids in the acidification products (Table 1). And the

298

analysis of acetate kinase and CoA transferase showed that both enzymes were increased by cysteine (Figure

299

3A). Moreover, both hydrogen and carbon dioxide were produced in the process of L-glucose acidification,

300

and pyruvate-ferredoxin oxidoreductase was increased by cysteine (Figure 3A). In addition, the produced

301

hydrogen and carbon dioxide were proved to be bio-converted to acetic acid via the participation of two key

302

enzymes (formate dehydrogenase and formyltetrahydrofolate synthetase) (Figures 3A and 3B). According to

303

these observations, the metabolic pathway for acetic and propionic acids production from L-glucose in the

304

presence of cysteine is proposed in Figure 4.

1200

45 Hydrogen (mL) or acetic acid (mgCOD/L）

A

1100

% of control

1000 900 200 150 100 50 0

LGDH

AK

CoAT

POR

FDH

FTHFS

Control

40

B

Cysteine

35

30

25

20

305 306

Figure 3. The influences of cysteine on activities of main enzymes involved in L-glucose acidification (A)

307

and hydrogen consumption and acetic acid production by homoacetogens (B). In the acidification test the

308

activities of LGDH, AK, CoAT, POR, FDH, and FTHFS were 0.0035±0.0011 (µM/min/mg-protein),

309

1.36±0.15 (U/mg-prote), 1.06±0.15 (U/mg-prote), 1.01±0.10 (U/mg-prote), 0.008±0.001 (U/mg-prote), and

310

4.5±0.3 (U/mg-prote), respectively, in the control reactor. Error bars represent standard deviations of

311

triplicate tests.

Hydrogen consumption

Acetic acid generation

312

As shown in Figure 4 pyruvic acid, an important metabolic intermediate, plays a vital role in

313

acidification of L-glucose. Since the biotransformation of L-glucose to L-glucono-1,5-lactone, catalyzed by

314

L-glucose dehydrogenase (LGDH), was reported to be the rate-controlled step when L-glucose was

315

metabolized to pyruvic acid,39 the improvement of LGDH would cause the increase of pyruvic acid, which

316

could finally enhance the production of acetic and propionic acids. Figure 3A showed that the enzyme

317

activity of L-glucose dehydrogenase was 10.9 folds of the control after L-cysteine addition, which was in

318

correspondence with the increased L-glucose degradation. Thus, more pyruvic acid could be generated, and

319

greater acetic and propionic acids would be produced, which was a key mechanism of cysteine in this study. 13



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320

In addition, it is well known that the contact between enzymes and substrates is the prerequisite for

321

achieving high efficient biological reactions. As cysteine was reported to promote the combination of

322

enzyme and substrate,54 its addition to L-glucose acidification system therefore caused the improvement of

323

L-glucose acidification. Although cysteine was reported to scavenge oxygen from reaction system,27 but all

324

current experiments were sparged with nitrogen gas for 5 min, and the influence of sparging time on

325

acidification was insignificant (p>0.05) (Table S4, Supporting Information), which suggested that the

326

saprging time reported in this study was enough to removal oxygen from the reactors, and cysteine did not

327

act as an oxygen scavenging in this study. It is well known that cysteine can be served as a nitrogen source

328

when the content of nitrogen is not enough for microbial activity, and the required nitrogen/carbon ratio

329

(TN/BOD) for microbes is usually in the range of 5/200 -5/100, i.e., 1/40 -1/20. In this study, however, the

330

ratios of TN/BOD in the carbohydrate addition tests were 1/(2.61-6.48), which were much greater than

331

1/(20-40). Thus, in the current study the production of methane enhanced by cysteine was not due to its

332

service as a nitrogen source. L-glucose

LGDH

L-glucono-1,5-lactone

L-gluconoate

L-5-ketogluconoate NADPH

NAD+ NADH

H2O

NAD+ NADH

LgnH

LgnG

LgnI

NADP+

ADP ATP

LgnE

GAP

LgnG

KDPGal

LgnF

KDGal

D-idonate

PTA

Pyruvic acid ATP ADP

Acetyl-CoA

Acetyl phosphate

Acetic acid AK

2NAD

Pi

2NADH

CoA

Oxaloacetic acid NADH 2FdH NAD+

2Fd

CO2

Malic acid POR

Fumaric acid FADH2

FDH & FTHFS

Hydrogen

FAD Succinic acid Succinyl-CoA

ADP ATP Methymalonyl-CoA

Propionyl-CoA

Propionic acid

CoA transferase

333 334

Figure 4. The proposed metabolic pathway for acetic and propionic acids produced from L-glucose in the

335

presence of cysteine. The key enzymes assayed in this study are labeled in red words. D-2-keto-3-

336

deoxygalactonate (KDGal), D-2-keto-3-deoxy-6-phosphogalactonate (KDPGal), D-glyceraldehyde-3-

337

phosphate (GAP), L-glucose dehydrogenase (LGDH), acetate kinase (AK), pyruvate-ferredoxin

338

oxidoreductase (POR), formate dehydrogenase (FDH), and formyltetrahydrofolate synthetase (FTHFS).

339

The generation of acetic acid and propionic acid is directly affected by the activities of AK of acetogen

14


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and CoAT of propionibacteria, respectively (Figure 4). Compared with the control, the data in Figure 3A

341

revealed that the activities of AK and CoAT were all significantly increased by cysteine addition (p

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