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Oxygen minimum zones are major sites of fixed nitrogen loss in the ocean. Recent studies have highlighted the importance of anaerobic ammonium oxidation, anammox, in pelagic nitrogen removal. Sources of ammonium for the anammox reaction, however, remain controversial, as heterotrophic denitrification and alternative anaerobic pathways of organic matter remineralization cannot account for the ammonium requirements of reported anammox rates. Here, we explore the significance of microaerobic respiration as a source of ammonium during organic matter degradation in the oxygen-deficient waters off Namibia and Peru. Experiments with additions of double-labelled oxygen revealed high aerobic activity in the upper OMZs, likely controlled by surface organic matter export. Consistently observed oxygen consumption in samples retrieved throughout the lower OMZs hints at efficient exploitation of vertically and laterally advected, oxygenated waters in this zone by aerobic microorganisms. In accordance, metagenomic and metatranscriptomic analyses identified genes encoding for aerobic terminal oxidases and demonstrated their expression by diverse microbial communities, even in virtually anoxic waters. Our results suggest that microaerobic respiration is a major mode of organic matter remineralization and source of ammonium (~45-100%) in the upper oxygen minimum zones, and reconcile hitherto observed mismatches between ammonium producing and consuming processes therein.

Nutrient measurements indicate that 30-50% of the total nitrogen (N) loss in the ocean occurs in oxygen minimum zones (OMZs). This pelagic N-removal takes place within only ~0.1% of the ocean volume, hence moderate variations in the extent of OMZs due to global warming may have a large impact on the global N-cycle. We examined the effect of oxygen (O(2)) on anammox, NH(3) oxidation and NO(3)(-) reduction in (15)N-labeling experiments with varying O(2) concentrations (0-25 µmol L(-1)) in the Namibian and Peruvian OMZs. Our results show that O(2) is a major controlling factor for anammox activity in OMZ waters. Based on our O(2) assays we estimate the upper limit for anammox to be ~20 µmol L(-1). In contrast, NH(3) oxidation to NO(2)(-) and NO(3)(-) reduction to NO(2)(-) as the main NH(4)(+) and NO(2)(-) sources for anammox were only moderately affected by changing O(2) concentrations. Intriguingly, aerobic NH(3) oxidation was active at non-detectable concentrations of O(2), while anaerobic NO(3)(-) reduction was fully active up to at least 25 µmol L(-1) O(2). Hence, aerobic and anaerobic N-cycle pathways in OMZs can co-occur over a larger range of O(2) concentrations than previously assumed. The zone where N-loss can occur is primarily controlled by the O(2)-sensitivity of anammox itself, and not by any effects of O(2) on the tightly coupled pathways of aerobic NH(3) oxidation and NO(3)(-) reduction. With anammox bacteria in the marine environment being active at O(2) levels ~20 times higher than those known to inhibit their cultured counterparts, the oceanic volume potentially acting as a N-sink increases tenfold. The predicted expansion of OMZs may enlarge this volume even further. Our study provides the first robust estimates of O(2) sensitivities for processes directly and indirectly connected with N-loss. These are essential to assess the effects of ocean de-oxygenation on oceanic N-cycling.

Metaproteomics has matured into a powerful tool to assess functional interactions in microbial communities. While many metaproteomic workflows are available, the impact of method choice on results remains unclear. Here, we carry out a community-driven, multi-laboratory comparison in metaproteomics: the critical assessment of metaproteome investigation study (CAMPI). Based on well-established workflows, we evaluate the effect of sample preparation, mass spectrometry, and bioinformatic analysis using two samples: a simplified, laboratory-assembled human intestinal model and a human fecal sample. We observe that variability at the peptide level is predominantly due to sample processing workflows, with a smaller contribution of bioinformatic pipelines. These peptide-level differences largely disappear at the protein group level. While differences are observed for predicted community composition, similar functional profiles are obtained across workflows. CAMPI demonstrates the robustness of present-day metaproteomics research, serves as a template for multi-laboratory studies in metaproteomics, and provides publicly available data sets for benchmarking future developments., (© 2021. The Author(s).)

Field studies are central to environmental microbiology and microbial ecology, because they enable studies of natural microbial communities. Metaproteomics, the study of protein abundances in microbial communities, allows investigators to study these communities " in situ ," which requires protein preservation directly in the field because protein abundance patterns can change rapidly after sampling. Ideally, a protein preservative for field deployment works rapidly and preserves the whole proteome, is stable in long-term storage, is nonhazardous and easy to transport, and is available at low cost. Although these requirements might be met by several protein preservatives, an assessment of their suitability under field conditions when targeted for metaproteomic analyses is currently lacking. Here, we compared the protein preservation performance of flash freezing and the preservation solution RNA later using the marine gutless oligochaete Olavius algarvensis and its symbiotic microbes as a test case. In addition, we evaluated long-term RNA later storage after 1 day, 1 week, and 4 weeks at room temperature (22°C to 23°C). We evaluated protein preservation using one-dimensional liquid chromatography-tandem mass spectrometry. We found that RNA later and flash freezing preserved proteins equally well in terms of total numbers of identified proteins and relative abundances of individual proteins, and none of the test time points was altered, compared to time zero. Moreover, we did not find biases against specific taxonomic groups or proteins with particular biochemical properties. Based on our metaproteomic data and the logistical requirements for field deployment, we recommend RNA later for protein preservation of field-collected samples targeted for metaproteomic analyses. IMPORTANCE Metaproteomics, the large-scale identification and quantification of proteins from microbial communities, provide direct insights into the phenotypes of microorganisms on the molecular level. To ensure the integrity of the metaproteomic data, samples need to be preserved immediately after sampling to avoid changes in protein abundance patterns. In laboratory setups, samples for proteomic analyses are most commonly preserved by flash freezing; however, liquid nitrogen or dry ice is often unavailable at remote field locations, due to their hazardous nature and transport restrictions. Our study shows that RNA later can serve as a low-hazard, easy-to-transport alternative to flash freezing for field preservation of samples for metaproteomic analyses. We show that RNA later preserves the metaproteome equally well, compared to flash freezing, and protein abundance patterns remain stable during long-term storage for at least 4 weeks at room temperature.

In the Western world, increased consumption of carbonated soft drinks combined with a decreasing intake of milk may increase the risk of osteoporosis. This study was designed to reflect the trend of replacing milk with carbonated beverages in a group of young men on a low-calcium diet and studies the effects of this replacement on calcium homeostasis and bone turnover. This controlled crossover intervention study included 11 healthy men (22-29 years) who were given a low-calcium basic diet in two 10-day intervention periods with an intervening 10-day washout. During one period, they drank 2.5 l of Coca Cola per day and during the other period 2.5 l of semi-skimmed milk. Serum concentrations of calcium, phosphate, 25-hydroxycholecalciferol, 1,25-dihydroxycholecalciferol (1,25(OH)2D), osteocalcin, bone-specific alkaline phosphatase (B-ALP) and cross-linked C-telopeptides (CTX), plasma intact parathyroid hormone (PTH) and urinary cross-linked N-telopeptides (NTX) were determined at baseline and endpoint of each intervention period. An increase in serum phosphate (P<0.001), 1,25(OH)2D (P<0.001), PTH (P=0.046) and osteocalcin (P<0.001) was observed in the cola period compared to the milk period. Also, bone resorption was significantly increased following the cola period, seen as increased serum CTX (P<0.001) and urinary NTX (P<0.001) compared to the milk period. No changes were observed in serum concentrations of calcium or B-ALP. This study demonstrates that over a 10-day period high intake of cola with a low-calcium diet induces increased bone turnover compared to a high intake of milk with a low-calcium diet. Thus, the trend towards a replacement of milk with cola and other soft drinks, which results in a low calcium intake, may negatively affect bone health as indicated by this short-term study.