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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 inhibitions as a percentage of growth rate without inhibitor subtracted from 100% at concentrations of each inhibitor causing approximately 20% inhibition H = 10 mM HMF, L = 5 mM furfural, A = 125 mM acetate, F = 50 mM formate. The dashed lines represent the inhibitory level if inhibitions were additive. 1) H; 2) L; 3) A; 4) F; 5) HL; 6) LA, 7) AF; 8) HA; 9) HF; 10) LF; 11) HLF; 12) HAF; 13) LAF; 14) HLA; 15) HLAF; 16-19) ½ of the levels of all compounds (HLAF).
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Figure 6: Growth inhibitions as a percentage of growth rate without inhibitor subtracted from 100% at concentrations of each inhibitor causing approximately 20% inhibition H = 10 mM HMF, L = 5 mM furfural, A = 125 mM acetate, F = 50 mM formate. The dashed lines represent the inhibitory level if inhibitions were additive. 1) H; 2) L; 3) A; 4) F; 5) HL; 6) LA, 7) AF; 8) HA; 9) HF; 10) LF; 11) HLF; 12) HAF; 13) LAF; 14) HLA; 15) HLAF; 16-19) ½ of the levels of all compounds (HLAF).

Mentions: The results are shown in Figure 6 as the percent of inhibition from the control without the inhibitor. The first four bars represent the percent inhibition caused by each of the four compounds individually: HMF (H), furfural (L), acetate (A) and formate (F), respectively. The presence of each compound caused ~ 20% growth inhibition, compared to the control, except for HMF which caused 28% inhibition of growth. The dashed line indicates theoretical levels of inhibition should the effect of the toxins be additive. For example, since the growth rate in HMF is 72% that of the control and in furfural, 81% of the control, the combination would result in 72% × 81% = 58% of the control growth rate, or 42% growth rate inhibition. Experimentally, we obtained 44% inhibition for the combination of HMF and furfural, indicating that both inhibitors did not act synergistically. In the presence of acetate and formate, we did see significant deviations from expected inhibitions, obtaining from 16-24% higher growth rate inhibitions with combinations of AF, HAF, LAF and HLAF. Results were also analyzed by Design Expert Version 7 from Stat-Ease, Inc. (Minneapolis, MN), which indicated that an interaction between acetate and formate is significant. (Additional file 4: Figure S4), however even though the model was significant, lack of fit was also noteworthy, indicating that more data points are needed to confirm this hypothesis.


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 inhibitions as a percentage of growth rate without inhibitor subtracted from 100% at concentrations of each inhibitor causing approximately 20% inhibition H = 10 mM HMF, L = 5 mM furfural, A = 125 mM acetate, F = 50 mM formate. The dashed lines represent the inhibitory level if inhibitions were additive. 1) H; 2) L; 3) A; 4) F; 5) HL; 6) LA, 7) AF; 8) HA; 9) HF; 10) LF; 11) HLF; 12) HAF; 13) LAF; 14) HLA; 15) HLAF; 16-19) ½ of the levels of all compounds (HLAF).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Growth inhibitions as a percentage of growth rate without inhibitor subtracted from 100% at concentrations of each inhibitor causing approximately 20% inhibition H = 10 mM HMF, L = 5 mM furfural, A = 125 mM acetate, F = 50 mM formate. The dashed lines represent the inhibitory level if inhibitions were additive. 1) H; 2) L; 3) A; 4) F; 5) HL; 6) LA, 7) AF; 8) HA; 9) HF; 10) LF; 11) HLF; 12) HAF; 13) LAF; 14) HLA; 15) HLAF; 16-19) ½ of the levels of all compounds (HLAF).
Mentions: The results are shown in Figure 6 as the percent of inhibition from the control without the inhibitor. The first four bars represent the percent inhibition caused by each of the four compounds individually: HMF (H), furfural (L), acetate (A) and formate (F), respectively. The presence of each compound caused ~ 20% growth inhibition, compared to the control, except for HMF which caused 28% inhibition of growth. The dashed line indicates theoretical levels of inhibition should the effect of the toxins be additive. For example, since the growth rate in HMF is 72% that of the control and in furfural, 81% of the control, the combination would result in 72% × 81% = 58% of the control growth rate, or 42% growth rate inhibition. Experimentally, we obtained 44% inhibition for the combination of HMF and furfural, indicating that both inhibitors did not act synergistically. In the presence of acetate and formate, we did see significant deviations from expected inhibitions, obtaining from 16-24% higher growth rate inhibitions with combinations of AF, HAF, LAF and HLAF. Results were also analyzed by Design Expert Version 7 from Stat-Ease, Inc. (Minneapolis, MN), which indicated that an interaction between acetate and formate is significant. (Additional file 4: Figure S4), however even though the model was significant, lack of fit was also noteworthy, indicating that more data points are needed to confirm this hypothesis.

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