pH indicator is used that turns the test solution red if the pH drops below 4.4. If the fermentation pathway has taken place, the mixture of acids it has produced will make the solution very acidic and cause a red colour change. The methyl red test belongs to a group known as the IMViC tests. Metabolic engineering Multiple bacterial strains have been metabolically engineered to increase the individual yields of end products formed by mixed acid fermentation. For instance, strains for the increased production of ethanol, lactate, succinate and acetate have been developed due to the usefulness of these products in biotechnology. The major limiting factor for this engineering is the need to maintain a redox balance in the mixture of acids produced by the fermentation pathway. For ethanol production Ethanol is the most commonly used biofuel and can be produced on large scale via fermentation. The maximum theoretical yield for the production of ethanol was achieved around 20 years. A plasmid that carried the pyruvate decarboxylase and alcohol dehydrogenase genes from the bacteria Z. mobilis was used by scientists. This was inserted into E. coli and resulted in an increased yield of ethanol. The genome of this E. coli strain, KO11, has more recently been sequenced and mapped. For acetate production The E. coli strain W3110 was genetically engineered to generate 2 moles of acetate for every 1 mole of glucose that undergoes fermentation. This is known as a homoacetate pathway. For lactate production Lactate can be used to produce a bioplastic called polylactic acid (PLA). The properties of PLA depend on the ratio of the two optical isomers of lactate (D-lactate and L-lactate). D-lactate is produced by mixed acid fermentation in E. coli. Early experiments engineered the E. coli strain RR1 to produce either one of the two optical isomers of lactate. Later experiments modified the E. coli strain KO11, originally developed to enhance ethanol production. Scientists were able to increase the yield of D-lactate from fermentation by performing several deletions. For succinate production Increasing the yield of succinate from mixed acid fermentation was first done by overexpressing the enzyme PEP carboxylase. This produced a succinate yield that was approximately 3 times greater than normal. Several experiments using a similar approach have followed. Alternative approaches have altered the redox and ATP balance to optimize the succinate yield. Related fermentation pathways There are a number of other fermentation pathways