In contrast, cysteine exposure reduced the biofilm biomass as effectively as HICA at acidic pH, although no reduction in metabolic activity was observed. Cysteine is vital for cellular function, but high levels cause accumulation of toxic sulfite. This is known to be toxic to bacteria and fungi but its impact on biofilms has not been studied. Although a very low concentration of ethanol was used in this study a reduction in biofilm biomass of 33% at pH 5.2 and 22% at pH 7.4 was observed. This is of clinical interest as ethanol is used in antifungal lock therapy against Candida spp. biofilms. In the human body ACH is metabolized into acetate mainly by the mitochondrial Ald enzyme. In our study, ALD5 was the only Ald enzyme encoding gene highly expressed in C. albicans biofilms in all conditions tested. C. albicans ALD5 is an ortholog of S. cerevisiae ALD5, which encodes the mitochondrial Ald enzyme. Previously our group showed that ALD6, encoding the cytosolic counterpart, is highly expressed in hypoxic conditions in planktonically grown cells and the expression correlated well with ACH levels. In early biofilms exposed to cysteine and PBS, a mild increase in the basal expression of ALD6 relative to the reference gene was observed thus supporting the finding of planktonic cultures. There was a correlation between ACH production and ALD5 expression in our study. Impairment of pyruvate bypass downstream metabolism by down-regulation of ALD5 was observed together with high ACH levels in caspofungin biofilms. ADH1 was also highly expressed but not in all conditions. On exposure to caspofungin, both ADH1 and ADH2 were down-regulated potentially resulting in ACH accumulation. This down-regulation correlates with the results of a previous study on 30 h C. albicans biofilms. The positive correlation between expression of ADH1 and ADH2 seen in this study implies a functional role of Adh2p alongside Adh1p. This finding is in line with previous work on planktonic cultures in hypoxia. The highest levels of ADH1 expression were observed after exposure to PBS, HICA and cysteine and mainly at acidic pH. Gene expression from PBS biofilms reflected an induction of fermentation and pyruvate bypass as many genes in the pathway were highly expressed. Considering the toxicity of HICA and cysteine particularly at acidic pH, the up-regulation of ADH1 and ALD5 could be a response to oxidative stress and impairment of other respiratory functions. Interestingly, upregulation of ADH1 has been noted in apoptosis. The significantly high basal expression of ADH1 and ALD5 in this study highlights their role in biofilms and supports the findings by others in vitro and in vivo. Alcohol abuse is the main etiologic agent in upper digestive tract carcinogenesis and multiple studies by our group and others have shown that the carcinogenic effect of alcohol is a result of microbial metabolism of ethanol to ACH in vitro and in vivo.