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Inhibition of growth of Zymomonas mobilis by model compounds found in lignocellulosic hydrolysates.

Franden MA, Pilath HM, Mohagheghi A, Pienkos PT, Zhang M - Biotechnol Biofuels (2013)

Bottom Line: An understanding of the toxic effects of compounds found in hydrolysate is critical to improving sugar utilization and ethanol yields in the fermentation process.Growth in xylose was profoundly inhibited by monocarboxylic organic acids compared to growth in glucose, whereas dicarboxylic acids demonstrated little or no effects on growth rate in either substrate.HMF (5-hydroxymethylfurfural), furfural and acetate, which were observed to contribute to inhibition of Z. mobilis growth in dilute acid pretreated corn stover hydrolysate, do not interact in a synergistic manner in combination.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Bioenergy Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA. mary.ann.franden@nrel.gov.

ABSTRACT

Background: During the pretreatment of biomass feedstocks and subsequent conditioning prior to saccharification, many toxic compounds are produced or introduced which inhibit microbial growth and in many cases, production of ethanol. An understanding of the toxic effects of compounds found in hydrolysate is critical to improving sugar utilization and ethanol yields in the fermentation process. In this study, we established a useful tool for surveying hydrolysate toxicity by measuring growth rates in the presence of toxic compounds, and examined the effects of selected model inhibitors of aldehydes, organic and inorganic acids (along with various cations), and alcohols on growth of Zymomonas mobilis 8b (a ZM4 derivative) using glucose or xylose as the carbon source.

Results: Toxicity strongly correlated to hydrophobicity in Z. mobilis, which has been observed in Escherichia coli and Saccharomyces cerevisiae for aldehydes and with some exceptions, organic acids. We observed Z. mobilis 8b to be more tolerant to organic acids than previously reported, although the carbon source and growth conditions play a role in tolerance. Growth in xylose was profoundly inhibited by monocarboxylic organic acids compared to growth in glucose, whereas dicarboxylic acids demonstrated little or no effects on growth rate in either substrate. Furthermore, cations can be ranked in order of their toxicity, Ca++ > > Na+ > NH4+ > K+. HMF (5-hydroxymethylfurfural), furfural and acetate, which were observed to contribute to inhibition of Z. mobilis growth in dilute acid pretreated corn stover hydrolysate, do not interact in a synergistic manner in combination. We provide further evidence that Z. mobilis 8b is capable of converting the aldehydes furfural, vanillin, 4-hydroxybenzaldehyde and to some extent syringaldehyde to their alcohol forms (furfuryl, vanillyl, 4-hydroxybenzyl and syringyl alcohol) during fermentation.

Conclusions: Several key findings in this report provide a mechanism for predicting toxic contributions of inhibitory components of hydrolysate and provide guidance for potential process development, along with potential future strain improvement and tolerance strategies.

No MeSH data available.


Related in: MedlinePlus

Growth rates for Z. mobilis 8b in RMG with increasing concentrations of inhibitor. A) ammonium levulinate, ammonium lactate, ammonium succinate; B) ammonium acetate, ammonium itaconate, ammonium 2-furoate and ammonium formate; C) potassium oxalate, furfuryl alcohol, ammonium vanillate and ammonium ferulate; D) ammonium caproate, ammonium 4-hydroxybenzoate, ammonium 4-hydroxycinnamate and ammonium benzoate; E) ammonium nitrate, ammonium hydrochlorate, ammonium sulfate and ammonium phosphate; F) syringaldehyde, vanilin,4-hydroxybenzaldehyde.
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Figure 1: Growth rates for Z. mobilis 8b in RMG with increasing concentrations of inhibitor. A) ammonium levulinate, ammonium lactate, ammonium succinate; B) ammonium acetate, ammonium itaconate, ammonium 2-furoate and ammonium formate; C) potassium oxalate, furfuryl alcohol, ammonium vanillate and ammonium ferulate; D) ammonium caproate, ammonium 4-hydroxybenzoate, ammonium 4-hydroxycinnamate and ammonium benzoate; E) ammonium nitrate, ammonium hydrochlorate, ammonium sulfate and ammonium phosphate; F) syringaldehyde, vanilin,4-hydroxybenzaldehyde.

Mentions: We used the Bioscreen C to determine growth rates for Z. mobilis 8b grown in the compounds listed in Table 1. All of these compounds have been identified, with the exception of oxalic acid, in dilute acid corn stover hydrolysates. Growth curves from cultures using glucose as the sole carbon source are shown in Figure 1 for each inhibitor concentration tested. Maximum growth rates, calculated after the cells doubled at least once within a 24 hour period, are plotted against the inhibitor concen-tration. The ammonium cation was used for each acid presented in Figure 1, with the exception of oxalic acid. Ammonium oxalate is insoluble in water above 50 mM at 30°C, therefore the potassium form was tested. Panels A, B, C and D display growth rate profiles for organic inhibitors and furfuryl alcohol are ranging from the least toxic organic acids in panel A to the most toxic in panel D to allow for better visualization of the data. Panels E and F show inhibition profiles of growth rate for Z. mobilis 8b grown in the presence of inorganic acids and three aldehydes, respectively. The growth curve profiles for HMF, furfural, and ethanol have already been reported [22]. Not surprisingly, ethanol was the least toxic compound studied. Z. mobilis is known for its high tolerance to ethanol. The parent strain, ZM4, has been shown to exhibit a similar specific growth rate when exposed to ethanol under continuous culture experiments [19].


Inhibition of growth of Zymomonas mobilis by model compounds found in lignocellulosic hydrolysates.

Franden MA, Pilath HM, Mohagheghi A, Pienkos PT, Zhang M - Biotechnol Biofuels (2013)

Growth rates for Z. mobilis 8b in RMG with increasing concentrations of inhibitor. A) ammonium levulinate, ammonium lactate, ammonium succinate; B) ammonium acetate, ammonium itaconate, ammonium 2-furoate and ammonium formate; C) potassium oxalate, furfuryl alcohol, ammonium vanillate and ammonium ferulate; D) ammonium caproate, ammonium 4-hydroxybenzoate, ammonium 4-hydroxycinnamate and ammonium benzoate; E) ammonium nitrate, ammonium hydrochlorate, ammonium sulfate and ammonium phosphate; F) syringaldehyde, vanilin,4-hydroxybenzaldehyde.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3716709&req=5

Figure 1: Growth rates for Z. mobilis 8b in RMG with increasing concentrations of inhibitor. A) ammonium levulinate, ammonium lactate, ammonium succinate; B) ammonium acetate, ammonium itaconate, ammonium 2-furoate and ammonium formate; C) potassium oxalate, furfuryl alcohol, ammonium vanillate and ammonium ferulate; D) ammonium caproate, ammonium 4-hydroxybenzoate, ammonium 4-hydroxycinnamate and ammonium benzoate; E) ammonium nitrate, ammonium hydrochlorate, ammonium sulfate and ammonium phosphate; F) syringaldehyde, vanilin,4-hydroxybenzaldehyde.
Mentions: We used the Bioscreen C to determine growth rates for Z. mobilis 8b grown in the compounds listed in Table 1. All of these compounds have been identified, with the exception of oxalic acid, in dilute acid corn stover hydrolysates. Growth curves from cultures using glucose as the sole carbon source are shown in Figure 1 for each inhibitor concentration tested. Maximum growth rates, calculated after the cells doubled at least once within a 24 hour period, are plotted against the inhibitor concen-tration. The ammonium cation was used for each acid presented in Figure 1, with the exception of oxalic acid. Ammonium oxalate is insoluble in water above 50 mM at 30°C, therefore the potassium form was tested. Panels A, B, C and D display growth rate profiles for organic inhibitors and furfuryl alcohol are ranging from the least toxic organic acids in panel A to the most toxic in panel D to allow for better visualization of the data. Panels E and F show inhibition profiles of growth rate for Z. mobilis 8b grown in the presence of inorganic acids and three aldehydes, respectively. The growth curve profiles for HMF, furfural, and ethanol have already been reported [22]. Not surprisingly, ethanol was the least toxic compound studied. Z. mobilis is known for its high tolerance to ethanol. The parent strain, ZM4, has been shown to exhibit a similar specific growth rate when exposed to ethanol under continuous culture experiments [19].

Bottom Line: An understanding of the toxic effects of compounds found in hydrolysate is critical to improving sugar utilization and ethanol yields in the fermentation process.Growth in xylose was profoundly inhibited by monocarboxylic organic acids compared to growth in glucose, whereas dicarboxylic acids demonstrated little or no effects on growth rate in either substrate.HMF (5-hydroxymethylfurfural), furfural and acetate, which were observed to contribute to inhibition of Z. mobilis growth in dilute acid pretreated corn stover hydrolysate, do not interact in a synergistic manner in combination.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Bioenergy Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO 80401, USA. mary.ann.franden@nrel.gov.

ABSTRACT

Background: During the pretreatment of biomass feedstocks and subsequent conditioning prior to saccharification, many toxic compounds are produced or introduced which inhibit microbial growth and in many cases, production of ethanol. An understanding of the toxic effects of compounds found in hydrolysate is critical to improving sugar utilization and ethanol yields in the fermentation process. In this study, we established a useful tool for surveying hydrolysate toxicity by measuring growth rates in the presence of toxic compounds, and examined the effects of selected model inhibitors of aldehydes, organic and inorganic acids (along with various cations), and alcohols on growth of Zymomonas mobilis 8b (a ZM4 derivative) using glucose or xylose as the carbon source.

Results: Toxicity strongly correlated to hydrophobicity in Z. mobilis, which has been observed in Escherichia coli and Saccharomyces cerevisiae for aldehydes and with some exceptions, organic acids. We observed Z. mobilis 8b to be more tolerant to organic acids than previously reported, although the carbon source and growth conditions play a role in tolerance. Growth in xylose was profoundly inhibited by monocarboxylic organic acids compared to growth in glucose, whereas dicarboxylic acids demonstrated little or no effects on growth rate in either substrate. Furthermore, cations can be ranked in order of their toxicity, Ca++ > > Na+ > NH4+ > K+. HMF (5-hydroxymethylfurfural), furfural and acetate, which were observed to contribute to inhibition of Z. mobilis growth in dilute acid pretreated corn stover hydrolysate, do not interact in a synergistic manner in combination. We provide further evidence that Z. mobilis 8b is capable of converting the aldehydes furfural, vanillin, 4-hydroxybenzaldehyde and to some extent syringaldehyde to their alcohol forms (furfuryl, vanillyl, 4-hydroxybenzyl and syringyl alcohol) during fermentation.

Conclusions: Several key findings in this report provide a mechanism for predicting toxic contributions of inhibitory components of hydrolysate and provide guidance for potential process development, along with potential future strain improvement and tolerance strategies.

No MeSH data available.


Related in: MedlinePlus