. Rillig and Antonovics (2019) have suggested that Closed Ecological Systems are particularly useful in understanding microbial interactions.
Laboratory microcosms lack many of the components of natural lakes (Carpenter 1996; Schindler 1998 ), but given the rich resources of archived lake data and its complexity of interpretation, it may be fruitful to examine whether phenomena seen in these CES studies occur in some lakes and not others. Earlier efforts to generalize lake ecology found common patterns in a Plankton Ecology Group (PEG) model that suggested many temperate lakes had an annual sequence of nutrient accumulation during winter, followed by a spring algal bloom, eliminated by a zooplankton bloom, which in turn was diminished by predation; controlling factors changed as the season progressed (Lampert and Sommer 2007; Sommer et al. 1986 ). The experiments described here conformed to the algal-grazer pattern. Tropical lakes may not experience an annual accumulation of nutrients with a predicted sequence, and flood lakes may have dynamics driven by wet/dry cycles; these might not show similar patterns.
The purpose of this study was to explore the O2 dynamics among a community of algae, grazers, and microbes under various nutrient regimens and isolated from atmospheric exchange.
Given a variety of initial nutrient conditions (e.g., C:N ratios), strong, constant patterns emerged that, at least superficially, seem similar to lake patterns. The consistency of the patterns suggested comparisons with archived lake data, though not an easy task, could increase our understanding of lake metabolism.
Organisms: Three species of green algae, Ankistrodesmus sp., Scenedesmus obliquus, and Selenastrum capricornutum, were grown separately in T82 medium (Astm 2012 )(see also Table S1 in Supplement). Daphnia magna were grown on these algae in a chemically defined medium, B/4 (Taub and Mclaskey 2014) ; see also Table S1. The algal cultures were unialgal, but they and the Daphnia cultures were not free of smaller microorganisms. Therefore, all respiration measurements included microbial processes as well as algae and grazers. Table S1 . In general, concentrations of ions are varied by factors of 4. The concentrations were slightly altered when 100 ml of mixed algal suspension in T82 medium was added (see Tables S1 and S2). These media approximate a modified Redfield Ratio (C 106: N 16: P 1) or normalized to nitrogen (C 6.625: N 1: P 0.0625), with the quantity of carbon being doubled because only approximately half of the carbon in NaHCO3 would be available for photosynthesis, the rest becoming CO3 -2 (Stumm and Morgan 1996) , which is not available to green algae (Falkowski and Raven 2007) . Phosphate was increased slightly to prevent it from being the limiting nutrient, and N:P ratios were kept constant.
Monitoring: Temperature, pressure, O2, pH, and conductivity were recorded each 5 minutes by a sonde (Manta2, Eureka Water Probes) inserted into a sealed culture container; the unit was mounted on a rotary shaker at 100-110 RPM. Total O2 concentrations for the liquid and gas phases were calculated (Taub and Mclaskey 2013) . The in vivo fluorescence of the settled algae was measured on a plant stress meter (PSM Mark II, BioMonitor SCI AB, Sweden). The
Daphnia were counted manually. More monitoring detail is provided in the Supplement.
Three findings stand out. The most surprising finding was that a period of intense grazing was associated with a diel O2 loss (negative NEP), which, if considered on a short-term basis, might be interpreted as heterotrophy, but was actually caused by the grazing of algae that had been produced earlier. Since these systems are closed to material exchanges, no organics could have entered. The reduced diel O2 production was the result of reduced algal populations. Given a reproducing Daphnia population, the algal density was often below our detection limits in many of our experiments. Our second finding of note is that small densities of algae, given available nutrients, could produce and use large amounts of O2, whereas denser, nutrientdepleted algae produced and used lessor amounts of O2. This could be a concern when community metabolism is estimated by multiplying organism density by a constant estimate of metabolism. Finally, there was strong linkage between O2 produced and O2 used; photosynthetically active communities had high respiration, whereas, once nutrients were sequestered, communities had both low photosynthetic and low respiratory rates.
A potential mechanism for the linkage between daytime and nighttime O2 changes could be that well-nourished fast-growing organisms have high metabolic rates and starved organisms often have low metabolic rates. For example, P-limited Selenastrum, one of our green algae, had a fivefold lower rate of respiration and a threefold reduction in CO2 fixation and O2 production, as compared with better nourished cells (Theodorou et al. 1991) , and Daphnia that had no or limited food for 15-20 hours increased their respiratory rates three-to fourfold when fed (Schmoker and Hernandez-Leon 2003) . There may be additional mechanisms that contribute to steady-state maintenance conditions, O2 nighttime uptake being similar to daytime O2 production. Even when photosynthesis has been minimized during nutrient deficiencies, the nighttime respiration was generally equal to or only slightly greater than the net produced the previous day.
Most findings conformed to expectations that a small number of algae inoculated into fresh medium would undergo a potential lag, exponential increase, stability, and decline (Fogg and Thake 1987) . The overgrazing of algae by Daphnia, and the eventual decline of the Daphnia and regrowth of algae, is also a familiar "predator-prey relationship" (Sommer et al. 1986 ). The data presented here are part of a larger series of experiments involving different C:N ratios (Taub 2019 ) and a variety of organic substrates (Taub 2009 ) with more experiments yet to be reported.
The shape of the diel O2 changes, with faster rates shortly after lights on and/or off, can have several interpretations. Sadro et al. (2011) and Schindler et al. (2017) have interpreted the rapid O2 loss at the beginning of darkness as microbial metabolism of photosynthetically produced dissolved organic carbon. Earlier studies interpreted increased respiration after photosynthesis as algal (or plant) metabolism of carbohydrates, later becoming slower as proteins are metabolized (Falkowski and Raven 2007) , but it is not certain if those samples were free of microbes. The shapes of both the rapid O2 increase at lights on and the rapid O2 decrease at lights off are similar to simple mathematical models of resource-dependent reactions, CO2 and possibly N and P being more abundant at lights on from metabolism during the previous night, and organic substrates being more abundant at the end of the lighted period and the beginning of darkness (lights off). The shapes are also similar to a more complex model involving production and destruction (recycling) (Odum and Odum 2000) .
Comparisons with photosynthesis-respiration (P-R) rates measured in natural aquatic communities almost all show a strong linear relationship with a lot of scatter (Solomon et al. 2013 ). Reasons for deviations from P approximating R could include an input of organic material that would increase respiration to exceed primary production or an input of inorganic nutrients that would increase photosynthesis above respiration.
Of greatest interest would be examining if intense grazing reduces diel O2 production and might be interpreted as heterotrophy (if short times are considered) but actually represents consumption of earlier photosynthetic products.
The CES experiments were originally designed to simulate the PEG model in which inorganic nutrients accumulate during a winter of low light intensity and temperature and spring brings an algal bloom that is terminated (in oligotrophic systems) by zooplankton grazing (Lampert and Sommer 2007; Sommer et al. 1986 ). Temperate lakes that conform to the PEG model might be likely to have algal-grazer dynamics similar to a Closed Ecological System. However, it is entirely possible that other factors may obscure metabolic activity, e.g., migrations, storms causing vertical mixing and terrestrial runoff, or flushing with upstream inputs and downstream drainage. Tropical lakes might not be expected to show behavior similar to that in a CES, since nutrients do not accumulate during a cold, dark period, leading to sequential blooms of phytoplankton, grazers, and predators; rather, wet and dry seasons may drive community responses. Flood lakes have episodic inputs of terrestrial materials that have major effects on community structure and metabolism (Holtgrieve et al. 2013; Holtgrieve et al. 2010 ).
Hypotheses based on simple systems might be used to explore data sets of naturally complex aquatic communities. In simple Closed Ecological Systems, there is a consistent linkage between daytime O2 increase and the following night's O2 decrease, with systems showing O2 levels will (1) gain, if nutrients and light are available; (2) maintain, with increases and decreases balancing (steady state); and (3) loss, after long periods of nutrient limitation. Do A.
B. Fig. 1 . An ungrazed Closed Ecological System consisting of mixed algae and associated microorganisms. A. increase in algal density (in vivo fluorescence); the initial value was too low to measure on the PSM instrument. B. Oxygen changes during 12:12 hour light/dark cycles with the exception of days 23-25 (8:16) and days 29-31 (18:6). Initially, O2 increased to a greater degree than it decreased in the dark with a net diel gain; later the increases and decreases were in close agreement, a period of maintenance. Note that all of the B figures have the same vertical axis of 500-2500 µmoles per ecosystem so they can be compared.
B. Algal abundance (in vivo fluorescence). The value at time 0 was too low to measure. B. Oxygen dynamics. Note the earlier period of gain, while daytime O2 production was greater than the following nighttime's loss (see changes in light/dark cycle days 10-15)* until the Daphnia grazing reduced the algal abundance, associated with decreases in total O2, the gain during the daytime being less than the following nighttime's loss. Eventually, the ecosystem maintained a fairly constant total O2 as the small daytime gains and nighttime losses were similar. *Short days 10-12, long days 13-15 (data lost for days 14 and 15). Medium was C 3.3, N 0.125, P 0.01 mM. 