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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

A) Aldehyde disappearance in shake flask fermentations of Z. mobilis 8b containing inhibitor at IC50 and B) corresponding growth curves monitoring absorbance for the following inhibitors: HMF (●),furfural (○),syringaldehyde (▼),vanillin (Δ) and 4-hydroxybenzaldehyde (■).
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Figure 7: A) Aldehyde disappearance in shake flask fermentations of Z. mobilis 8b containing inhibitor at IC50 and B) corresponding growth curves monitoring absorbance for the following inhibitors: HMF (●),furfural (○),syringaldehyde (▼),vanillin (Δ) and 4-hydroxybenzaldehyde (■).

Mentions: We observed the reduction of HMF and furfural by Z. mobilis during earlier fermentation studies conducted in our laboratory which has been previously reported [13]. High cell density inocula could improve fermentation performance by reducing the impact of toxic compounds, particularly if compounds were converted to a less toxic compound or intermediates. We evaluated compound conversions in shake flasks, using a high cell inoculum at concentrations of aldehydes, which cause 50% of growth inhibition. The concentrations of HMF, furfural, syringaldehyde, vanillin and 4-hydroxybenzaldehyde used for this experiment were 21.4, 15.6, 17.6, 4.3, and 5.7 mM, respectively. After 24 hours, all the sugar was consumed and most cultures reached stationary phase (Figure 7B) at final cell densities lower than the control (Figure 7B). After 48 hours, 97% of HMF or furfural had disappeared (Figure 7A). Surprisingly, Zymomonas also has the ability to metabolize most of 4-hydroxybenzaldehyde (95%) and 60% of vanillin after 48 hours. In control flasks without cells, most of the compounds remained at initial levels after 48 hours, except for vanillin and syringaldehyde which showed a loss of 14-16% over 48 hours, respectively. The amount of syringaldehyde removed with cells was at 10%, less than the abiotic control. The near-complete elimination of HMF, furfural, and 4-hydroxybenzaldehyde and partial elimination of vanillin occurred simultaneously with the appearance of single unique peaks on UV chromatograms (Additional file 1: Figure S1). The product of furfural conversion eluted at the same retention time and had the same spectral profile in the UV range (between 230 nm – 340 nm) as furfuryl alcohol. Likewise, the apparent conversion products of vanillin and 4-hydroxybenzaldehyde eluted at the same retention times and had the same spectral profile as vanillyl and 4-hydroxybenzyl alcohol. Confirmatory evidence that the conversion products were the alcohol analog of the added aldehydes was provided by GC-MS analysis. (Additional file 2: Figure S2). The conversion of the aldehyde to its alcohol form implies a reductive mechanism for conversion. Since the alcohol form of HMF is not commercially available, nor the mass spectrum available for this compound, we could not conclude that a reductive conversion pathway was operating for this compound, though it seems quite likely.


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)

A) Aldehyde disappearance in shake flask fermentations of Z. mobilis 8b containing inhibitor at IC50 and B) corresponding growth curves monitoring absorbance for the following inhibitors: HMF (●),furfural (○),syringaldehyde (▼),vanillin (Δ) and 4-hydroxybenzaldehyde (■).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: A) Aldehyde disappearance in shake flask fermentations of Z. mobilis 8b containing inhibitor at IC50 and B) corresponding growth curves monitoring absorbance for the following inhibitors: HMF (●),furfural (○),syringaldehyde (▼),vanillin (Δ) and 4-hydroxybenzaldehyde (■).
Mentions: We observed the reduction of HMF and furfural by Z. mobilis during earlier fermentation studies conducted in our laboratory which has been previously reported [13]. High cell density inocula could improve fermentation performance by reducing the impact of toxic compounds, particularly if compounds were converted to a less toxic compound or intermediates. We evaluated compound conversions in shake flasks, using a high cell inoculum at concentrations of aldehydes, which cause 50% of growth inhibition. The concentrations of HMF, furfural, syringaldehyde, vanillin and 4-hydroxybenzaldehyde used for this experiment were 21.4, 15.6, 17.6, 4.3, and 5.7 mM, respectively. After 24 hours, all the sugar was consumed and most cultures reached stationary phase (Figure 7B) at final cell densities lower than the control (Figure 7B). After 48 hours, 97% of HMF or furfural had disappeared (Figure 7A). Surprisingly, Zymomonas also has the ability to metabolize most of 4-hydroxybenzaldehyde (95%) and 60% of vanillin after 48 hours. In control flasks without cells, most of the compounds remained at initial levels after 48 hours, except for vanillin and syringaldehyde which showed a loss of 14-16% over 48 hours, respectively. The amount of syringaldehyde removed with cells was at 10%, less than the abiotic control. The near-complete elimination of HMF, furfural, and 4-hydroxybenzaldehyde and partial elimination of vanillin occurred simultaneously with the appearance of single unique peaks on UV chromatograms (Additional file 1: Figure S1). The product of furfural conversion eluted at the same retention time and had the same spectral profile in the UV range (between 230 nm – 340 nm) as furfuryl alcohol. Likewise, the apparent conversion products of vanillin and 4-hydroxybenzaldehyde eluted at the same retention times and had the same spectral profile as vanillyl and 4-hydroxybenzyl alcohol. Confirmatory evidence that the conversion products were the alcohol analog of the added aldehydes was provided by GC-MS analysis. (Additional file 2: Figure S2). The conversion of the aldehyde to its alcohol form implies a reductive mechanism for conversion. Since the alcohol form of HMF is not commercially available, nor the mass spectrum available for this compound, we could not conclude that a reductive conversion pathway was operating for this compound, though it seems quite likely.

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