Inhibitors

Inhibitors based on their chemical functional groups can be grouped as aldehydes, ketones, phenols, and organic acids.

  • Aldehyde inhibitors: compounds with one or more functional aldehyde groups, regardless of the base structure of a furan ring, a benzene ring or a phenol-related structure, including
      o furfural and HMF
           Cell walls and membranes of yeast cells grown under furfural and HMF-challenged conditions appear damaged when compared to those of controls grown in the absence of any inhibitor, resulting in the delay of cell growth and reduction of ethanol productivity
           Furfural and HMF inhibit cell growth and ethanol production rates at lower concentrations. Individual strains have been isolated that retain their ability to produce ethanol in the presence of 10 to 79 mM of either furfural or HMF, including strains of these species: Saccharomyces cerevisiae, Pichia stipitis, Candida shehatae, Corynebacterium glutamicum, Zymomonas mobilis, and Escherichia coli.
      o 4-hydroxybenzaldehyde, vanillin, syringaldehyde, isovanillin,ortho-vanillin, Cinnamaldehyde
         more inhibitory than those derived from sugar dehydration

• Ketone inhibitors:4-hydroxyacetopheone, acetovanillone and acetocsyringone
         exert a greater inhibitory effect on bacteria such as Thermoanaerobacter mathranii than on yeasts, in terms of reduced growth and ethanol yield

• Organic acid inhibitors: sharing a common carboxylic acid functional group, all contain a carboxyl functional group such as
       o acetic acid,formic acid, levulinic acid, caproic acid,furoic acid, 4-hydroxybenzoic acid, 3-hydroxybenzoic acid, 2-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, protocatechic acid, vanillic acid, gallic acid, syringic acid, 4-hydroxycinnamic acid, homovanillic acid, guaiaclyglycolic acid, and sinapic acid. These inhibitors are thought to be exert their inhibitory actions via their carboxyl functional groups.
             more toxic to isolates of bacteria than yeasts at low concentration
             The toxicity has been correlated with their degree of hydrophobicity, suggesting the involvement of a hydrophobic target such as the cell membrane

• phenol-based inhibitors: phenol, benzene-1,2-diol (catechol) etc.
      o cause increased membrane fluidity and affect membrane permeability, which may enhance synergistic inhibition when combined.
      o Phenols such as cathecol, hydroquinone, and coniferyl alcohol almost completely inhibit E. coli , but are relatively less toxic to yeast. However, eugenol and isoeugenol are inhibitory to yeasts at low concentrations.
     o The three main phenol structure building blocks in lignin show the order of inhibitory effects, ranked from strong to weak, as (1) hydroxyphenol, (2) guaiacyl, and (3) syringyl

In general, aldehydes and phenols are more toxic than organic acids.

In addition, some inhibitions will be amplified through process. A typical example is that fermentative microorganisms will be inhibited by concentrated non-volatiles such as lignin derivatives and extractives after concentrating although low initial inhibitor concentration in hydrolyzate.

Mechanism of furfural inhibition

• Furfural is metabolized by S. cerevisae under aerobic, oxygen-limited, and anareobic conditions to furfural alcohol.
  –Furfural reduce the specific growth rate, the cell-mass yield on ATP, the volumetric, and specific ethanol production
  –NADH-dependent yeast alcohol dehydrogenase (ADH) is believe to be responsible for furfural reduction.
  –Under anaerobic conditions, glycerol is normally produced to regenerate excess NADH formed in biosynthesis. Glycerol production reduction during furfural reduction suggests that furfural reduction regenerates NAD+.
  –Elevated concentrations of acetaldehyde excreted in the beginning of the fermentation, which was suggested to be due to a decreased NADH concentration in the cell during furfural reduction.
  –Furfural inhibition of glycolytic enzymes in vitro and the direct inhibition of ADH might have contributed to acetaldehyde excretion.
  –Intracellular acetaldehyde acumination suggested to be the reason for lag-phase in growth in the presence of furfural.
  –The model
  »i) furfural reduction to furfural alcohol by NADH dependent dehydrogenases had a higher priority than reduction of dihydroxyacetone phosphate to glycerol
  »ii) furfural caused inactivation of cell replication.

Mechanism of acidic acid inhibition

Uncoupling and intracellular anion acumination
–The drop in intracellular pH resulting from inflow of weak acids is neutralized by the action of plasma membrane ATPase, which pumps protons out of the cell at the expense of ATP hydrolysis. At high acid concentration, the proton pumping capacity of the cell is exhausted, resulting depletion of the ATP content, dissipation of the proton motive force, and acidification of the cytoplasm.
–However, the anionic forms of acetic, formic acid, are lipophobic and do not traverse the plasma membrane in both dissociated and undissociated form, causing a high rate of proton impart. The extent of intracellular anion accumination will be a function of the pH gradient over the plasma membrane.