Yeast chronological aging has a multifactorial nature. Many cellular processes and extrinsic factors negatively influence the CLS. Examples include oxidative stress, reduced autophagy or medium acidification. Processes which induce stress responsive genes extend yeast lifespan 
. Hence yeast CLS is determined by the resultant of multiple positive and negative processes. Because of this complexity several factors implicated in yeast aging are still highly debated.
So far most research on yeast CLS is performed with S. cerevisiae using glucose/ammonium sulphate containing media. In this paper we analysed the chronological lifespan of the yeast H. polymorpha in relation to growth on different carbon and nitrogen sources.
Our data revealed that of the three carbon sources tested (glucose relative to two compounds that require peroxisome function for growth namely ethanol and methanol) H. polymorpha
shows the shortest chronological lifespan on glucose. Compared to S. cerevisiae
, H. polymorpha
dies relatively fast with a short maximum lifespan of less than 4 days when grown on 0.5% glucose. At these conditions the maximum lifespan of S. cerevisiae
is generally above 10 days .
Glucose metabolism involves glycolysis, which in S. cerevisiae
leads to acetic acid formation that is associated with induction of the mitochondrial apoptosis pathway 
. In S. cerevisiae
acetic acid production is strongly reduced upon growth on glycerol instead of glucose 
(. Similar mechanisms most likely operate in H. polymorpha
, because the medium of glucose cultures acidified more strongly relative to those containing ethanol- or methanol. Although placing the glucose-grown cells in fresh buffer significantly extended the lifespan, neither the median nor maximum lifespan reached values obtained for methanol or ethanol cultures (). One explanation may be that prior to placing the glucose-grown cells in buffer, they already experienced the toxic effects related to the low pH when reaching the stationary phase.
In addition to medium acidification, ROS are important factors in determining yeast CLS. ROS initially were assumed to be harmful as they caused oxidative damage. However, data have been presented indicating that ROS also can have a positive effect as signaling molecules that induce stress responsive genes (hormesis). Moreover, recent findings suggest that in S. cerevisiae
also the type of ROS (e.g. superoxide versus hydrogen peroxide) and the growth stage at which they occur are important for lifespan extension 
, which illustrates a complex role of ROS in yeast aging.
We observed that in H. polymorpha
ROS levels were equally low during the first days of the CLS experiments in glucose, ethanol and methanol cultures. Because in this period differences in survival were already evident, ROS levels alone most likely are not the major determinants in the observed differences in lifespan. At later stages ROS levels increased in the methanol and ethanol cultures. Given the multifactorial nature of CLS, it is yet unclear whether this may have caused negative and/or positive effects. Also, ROS measurements performed with fluorescent dyes have to be interpreted with care when cells are grown on different carbon sources. We used DHR, which forms fluorescent rhodamine efficiently upon reaction with free •
OH or NO2•
radicals, but requires a catalyst for oxidation by O2•
. Important catalysts are iron, heme and cytochrome c oxidase. These catalysts as well as the composition of the ROS may vary significantly upon growth of H. polymorpha
cells on the different carbon sources. For instance, the peroxisomal heme containing enzyme catalase is strongly induced on methanol, to moderate levels on ethanol but repressed on glucose 
. Hence, relative to glucose heme levels and most likely also iron (e.g. released from catalase in aged cells) are may be significantly higher in methanol and ethanol cells which may add to the observed increase in ROS levels. Together, our data lend support to the view that cultivation of cells at conditions that require peroxisomes for growth is beneficial for the lifespan of the cells.
It cannot be excluded that other factors also contribute to the short CLS of glucose-grown H. polymorpha
. For instance, by-products of glycolysis like methylglyoxal were described to have a negative impact on cell survival 
. Further studies are required to fully dissect all factors involved.
Our data indicate that the presence of methylamine (MA) as sole nitrogen source instead of ammonium sulphate resulted in a significant extension of the chronological lifespan of methanol-grown cells. Because no differences in pH values were observed, medium acidification is not a major factor in the observed lifespan differences.
Amines have been described to universally enhance the lifespan of various models. For instance, spermidine acts as an anti-aging compound by inducing autophagy 
. Because we also observed the positive effect of MA on viability in H. polymorpha
cells that are defective in autophagy (), MA is unlikely to alter autophagy processes in this yeast species. Moreover, based on electron microscopy studies we did not obtain any morphological indications that MA induces or reduces autophagy in wild-type cells (data not shown).
Our studies indicate that MA oxidation by AMO and the subsequent generation of extra NADH, is an important reason for the lifespan extension by MA. We show that the positive effects of MA do not occur in an AMO deficient strain or when MA is removed from the medium, but occur again when formaldehyde, the oxidation product of MA, was added to the stationary phase cultures. This led us to conclude that the generation of additional NADH in carbon starved cells can postpone cell death by providing energy and reducing the intracellular environment. Indeed, our data () suggest that NADH generation due to methylamine metabolism changes the intracellular redox balance in the cell leading to lower ROS levels. Hence, the relatively high ROS levels observed in methanol/AS cultures (see also ) might present a detrimental effect.
Similar to MA, D-alanine can also be used as a nitrogen source by H. polymorpha
. Oxidative deamination of D-alanine generates pyruvate and ammonia. We anticipate that production of pyruvate during chronological aging generates energy and extends the CLS in similar fashion as MA 
Summarizing, our data are consistent with the view that multiple factors may be involved in lifespan extension caused by MA. Our data indicate that production of NADH generated from MA metabolism is the major factor. NADH contributes to ATP generation but also to reducing ROS. Additional factors may be involved as well, such as toxicity of ammonium in methanol/AS cultures, like recently reported for S. cerevisiae
Chronological aging of yeast cells has been proposed as a model for the post-mitotic cells in higher eukaryotes 
, a situation which is obviously dissimilar to starving cells in yeast stationary phase cultures. However, the use of an additional NADH generation system, which does not support growth, can make findings of yeast systems more applicable as model for higher eukaryotic cells.