Although glaciers have previously been shown to export pre-aged DOC that is highly bioavailable. The actual age of glacier DOC consumed by microbial communities has not been identified. This is significant as it has direct implications for atmospheric carbon budgets and positive feedbacks to climate warming.

Abstract

Add “bulk” before….”DOC",  to read "Relative to bulk DOC, ..."

Remove the “+” before 4,350

Introduction

Line 48- The phrasing of “…whether the aged component of glacier DOC is responsible for its high bioavailability” should be changed as it implies that the age determines its bioavailability, which is not the case as other chemical, physical and biological parameters determine the portion consumed. Perhaps something along the lines of “whether the aged component contributes to the bioavailable fraction”.

Line 60- delete “bioavailable” after “…the age and source of respired, bioavailable OC”

Methods

Line 75-82- How long was the filtrate stored prior to incubations? What was the filtrate stored in and what volume? The filtrate for respiration experiments was acidified and frozen?  Did this result in flocculation of DOC when thawed for these incubations and measurements? If so what fraction was lost as flocculant?

Line 87- How was the initial stripping of DIC from the incubation water verified?  Was DIC or pCO2 measured to assure complete removal?

Line 88- how supersaturated where the incubations with O2, that is an extremely high rate of O2 flow and duration for a 1L incubation.   It would have been ideal to return to initial O2 conditions. Supersaturation of O2 can significantly impair microbial decomposition due to formation of reactive oxygen species, interference with enzymes etc. What were the O2 levels at the time of incubation initiation?

Line 100- How was the evolved CO2 collected prior to transfer to the vacuum line?  Cryogenically?  The flow rate of 0.8  L min^-1 is much too fast for quantitative trapping. For example, Beaupre et al. 2007 (L&O methods) used a flow rate of  200ml min^-1. The 0.8  L min^-1 means that only a portion of the respired C would be captured, with a significant portion expelled from the Liq N2 trap, resulting in isotopic fractionation.  Thus the isotopic values measured do not reflect the signature of respired C, but only whatever fraction which was trapped and recovered.  How were these flow rates measured?

The experiment is lacking controls.  For example, a killed control, or glacial water than remains acidified and uninoculated and is allowed to sit for the 28 days.  How can you be sure that atmospheric CO2 was not introduced during the incubation, processing and harvesting, thereby skewing the value with modern CO2.  It does not look that UHP He or O2 was used, nor were traps added to potentially trap any residual CO2 in these sparge gases which also might contribute to the recovered CO2 signature.  Quantitative recovery is critical to ensure isotopic fidelity.  Documented quantitative recovery could be accomplished by adding in DIC with of a known quantity and isotopic signature and measuring the percent recovery and isotopic signature of the product. Currently it is impossible evaluate the validity of the reported respiratory CO2 isotopic signatures.    

Results and Discussion

What amount of respiratory CO2 was captured for each incubation?  Using that number, the percentage of bioavailable OC could be calculated.  How does this compare to past published results? One can assume on 28-day timescales that BGE is low and that the majority of DOC consumed is fueling respiration rather than biomass production.  But the authors should be careful to note this in their discussion, and elsewhere..that the portion that is respired is only a portion (though likely the majority) of bioavailable OC.

Lines 157-158- I would indicate that this is a two-endmember model and that it is assumed that all of the radiocarbon dead material is derived from fossil fuel combustion byproducts.

Line 184-186 and Figure 4- The trend of enriched d13C and younger CO2 with % RA of aliphatic compounds is driven largely by one point (supraglacial stream) out of 4.  I think these statements should be better qualified to recognize the limitations in the dataset.

Line 186-187-I disagree with the authors’ interpretation “This tendency suggests the 13C and 14C enriched source respired during the incubations may be relatively more abundant in the more aliphatic samples.” Instead doesn’t this tendency suggest that the 13C and 14C enriched source which is respired contains a greater percentage of aliphatic OC?

Line 197- The significant figures for d13C here and else where in the manuscript should be consistent and likely to the second decimal place, but to the first decimal place at a minimum.  

Line 200-201- Change “were postulated to reflect greater in situ produced over atmospherically deposited OC (Schmidt et al., 2022)” to “were postulated to reflect a greater contribution of in situ produced OC relative to atmospherically deposited OC” or something similar to clarify the meaning.

Line 206-211- “Additionally, we observe relative similarity in molecular and isotopic composition (DOC and respiratory CO2) between the supraglacial stream and glacier outflows, supporting a component of the bioavailable organics exported from these glaciers being derived from recent in situ production on the surface, rather than a subglacial source (i.e., since the outflow and supraglacial samples are relatively comparable there is little evidence for significant subglacial input of OC; Table 1, Figure 2 & 3). This sentence needs to be more concise or broken into two sentences.

Line 225-226- Please clarify “the observed 13C-CO2 enrichment could have stemmed from a 13C enriched fossil fuel source or compounds within the sources OC pool, or result from photodegradation processes (Spencer et al., 2009)”.  I am confused as to how to reconcile the isotopic differences based on this explanation.

Table 1 lacks uncertainties for all measurements.  Duplicates for DOC and d13C could have been easily run.  In addition, for the D14C respiration measurements, we have no assessment of error or reproducibility.  I recognize the cost of D14C measurements, but at least one of the samples should have been replicated to provide an error associated with the respiratory CO2 measurement. Further, what is the blank associated with the measurement?   The introduction of atmospheric CO2 during the incubation and CO2 harvest could have easily shifted the respiratory CO2 to the more modern and d13C enriched (~ -7 per mil) values.  Without a process blank one cannot back out isotopic artefacts from the experimental treatment.  In addition, there are no CO2 recovery values.  These amounts could be used to compare recoveries to previous C consumption in bioassays to see if they are realistic.  They could also be employed to run some back of the envelope calculations on the bulk DOC to see whether it is feasible for x portion of a C13 enriched pool to be hidden.

General comments:

This paper uses multiple analytical techniques to characterize the pool of dissolved organic carbon in the outflow of three Alaskan Glaciers, and in a supraglacial stream of one of those glaciers. The authors pair these measurements of glacial DOC with bioincubation experiments and measure the isotopic signature of respired CO_2. They conclude that the young and aliphatic-rich portion of the DOM pool is most bioavailable. The paper is generally well written, and the suite of measurements provide valuable insights into glacially-sourced DOM cycling, which is of appropriate scope for The Cryosphere. However, there are questions/comments detailed below regarding conclusions drawn from this experimental design, which lacks controls, replicates, and measures of uncertainty. These issues should be addressed, and conclusions carefully articulated within the limitations of the dataset.

Specific Comments:

Figure 1: this figure could be significantly improved so that each glacier is visible. Consider using a satellite image (from Jul, or ideally close to the field sampling period) and zoom in enough so that precise sampling locations are visible relative to the glacier terminus/proglacial lake/stream network. 

Section 2.1: This section would benefit from a more detailed description of these glaciers, including comments on their potential subglacial hydrology and geology, and biogeochemical context from previous work.

Line 77: provide more details on where the supraglacial sample was collected. Improvements to Figure 1 will help, but it’s worth noting here that it came from a supraglacial stream (which you mention in results). Knowing where was collected relative to the terminus/snow line has implications for interpreting DOC sources.

Line 77: describe where you collected your outflow samples in each catchment. This is especially important for Mendenhall since it terminates in a proglacial lake. Understanding the location relative to local/micro-environments and non-glacial stream influence is relevant to water/DOC source, and residence/transit times. This would also be a good spot to describe the timing of your sample collection in relation to the seasonal evolution of the glacial hydrology and ultimately theoretical water/DOM sources. Do you expect the outflow at this time of year to represent an efficient drainage system with little influence from subglacial sources? If so, that means this paper is not really looking at the ‘bulk glacier DOM pool’ as the title suggests, and is instead looking at the lability of predominantly supraglacial DOM.

Table 1: report an indicator of uncertainty (including detection limits) for these analyses, here or elsewhere in the document.   This is particularly important for the instances where you compare supraglacial DOM to outflow DOM.

Figure 2: (a) and (b) are unnecessary since the same data are presented more efficiently in (c)

Section 3.2, line 157, line 210: given the increasing evidence for subglacial chemotrophic pathways, it’s worth commenting on the likelihood of these sources of DOM to the outflows, especially whether the subglacial environment may be a source of old but labile DOM. Additional information on the subglacial hydrology/geology in section 2.1 will help frame whether these systems are conductive to chemotrophic DOM production. Are there previous studies at your sites that would justify ruling this source out? Perhaps this is what you’re referring to in line 228, but I think it warrants a more thorough discussion than the statement in brackets on line 209-210.

Line 184-187: Without error bars or statistical analysis (Figure 4), I’m not sure you can claim these associations are significant or therefore make a justified link between the 13C and 14C signatures to the aliphatic content. Further, since the DOM characterization is not truly quantitative, RA was not measured before and after the incubation, and you do not present CO2 concentrations or quantify the DOC consumed during the incubation, I think it’s a stretch to claim that “the 13C and 14C enriched source respired during the incubation may be relatively more abundant in the more aliphatic samples” and then extend that further in the abstract to say “Molecular-level analyses suggest respired OC was associated with the aliphatic-rich portion of the DOM pool”.

Section 3.4: Do you know that there were no inorganic processes affecting CO2 concentrations/isotopic signatures, despite the 0.45 um filter? Glacial flour can have an ultra-fine component with high surface area, so it's theoretically possible.  It appears you did not collect CO2 concentrations or DOC after incubation, which is a shame since that would allow for a simple mass balance calculation and help isolate/confirm the process(es) at play. An experimental control would have also addressed this issue.  In the absence of these data and a robust experimental design with controls, I think you need to somehow make your case that inorganic processes and/or contamination are not contributing CO2 during incubation.