Continuous, real-time monitoring of the oxygen uptake rate (OUR) in animal cell bioreactors

A new method for real-time monitoring of the oxygen uptake rate (OUR) in bioreactors, based on dissolved oxygen (DO) measurement at two points, has been developed and tested extensively. The method has several distinct advantages over known techniques.It enables the continuous and undisturbed monitoring of OUR, which is conventionally impossible without gas analyzers. The technique does not require knowledge of k(L)a. It provides smooth, robust, and reliable signal. The monitoring scheme is applicable to both microbial and mammalian cell bioprocesses of laboratory or industrial scale. The method was successfully used in the cultivation of NSO-derived murine myeloma cell line producing monoclonal antibody. It was found that while the OUR increased with the cell density, the specific OUR decreased to approximately one-half at cell concentrations of 16 x 10(6) cells/mL, indicating gradual reduction of cell respiration activity. Apart from the laboratory scale cultivation, the method was applied to industrial scale perfusion culture, as well as to processes using other cell lines. (c) 1994 John Wiley & Sons, Inc.     

The effect of dissolved oxygen tension and the utility of oxygen uptake rate in insect cell culture

Dissolved oxygen tension and oxygen uptake rate are critical parameters in animal cell culture. However, only scarce information of such variables is available for insect cell culture. In this work, the effect of dissolved oxygen tension (DOT) and the utility of on-line oxygen uptake rate (OUR) measurements in monitoring Spodoptera frugiperda (Sf9) cultures were determined. Sf9 cells were grown at constant dissolved oxygen tensions in the range of 0 to 30%. Sf9 metabolism was affected only at DOT below 10%, as no significant differences on specific growth rate, cell concentration, amino acid consumption/production nor carbohydrates consumption rates were found at DOT between 10 and 30%. The specific growth rate and specific oxygen uptake rate followed typical Monod kinetics with respect to DOT. The calculated _max and max were 0.033 h^-1 and 3.82×10^-10 mole cell^-1h^-1, respectively, and the corresponding saturation constants were 1.91 and 1.57%, respectively. In all aerated cultures, lactate was consumed only after glucose and fructose had been exhausted. The yield of lactate increased with decreasing DOT. It is proposed, that an apparent DOT in non-instrumented cultures can be inferred from the lactate yield of bioreactors as a function of DOT. Such a concept, can be a useful and important tool for determining the average dissolved oxygen tension in non-instrumented cultures. It was shown that the dynamic behavior of OUR can be correlated with monosaccharide (fructose and glucose) depletion and viable cell concentration. Accordingly, OUR can have two important applications in insect cell culture: for on-line estimation of viable cells, and as a possible feed-back control variable in automatic strategies of nutrient addition.Abbreviations DOT Dissolved oxygen tension - OUR Oxygen uptake rate - specific oxygen uptake rate - specific growth rate - X_v viable cell concentration - C_L, C^*, and oxygen concentrations in liquid phase, in equilibrium with gas phase, and medium molar concentration, respectively - H Henry's constant - K_La volumetric oxygen transfer coefficient - P_T total pressure - oxygen partial pressure - oxygen molar fraction - i discrete element     

The effect of land use on dissolved organic carbon and nitrogen uptake in streams

1. Agricultural and urban land use may increase dissolved inorganic nitrogen (DIN) concentrations in streams and saturate biotic nutrient demand, but less is known about their impacts on the cycling of organic nutrients. To assess these impacts we compared the uptake of DIN (as ammonium, NH₄⁺), dissolved organic carbon (DOC, as acetate), and dissolved organic nitrogen (DON, as glycine) in 18 low‐gradient headwater streams in southwest Michigan draining forested, agricultural, or urban land‐use types. Over 3 years, we quantified uptake in two streams in each of the three land‐use types during three seasons (spring, summer and autumn). 2. We found significantly higher NH₄⁺ demand (expressed as uptake velocity, V_f) in urban compared to forested streams and NH₄⁺V_f was greater in spring compared to summer and autumn. Acetate V_f was significantly higher than NH₄⁺ and glycine V_f, but neither acetate nor glycine V_f were influenced by land‐use type or season. 3. We examined the interaction between NH₄⁺ and acetate demand by comparing simultaneous short‐term releases of both solutes to releases of each solute individually. Acetate V_f did not change during the simultaneous release with NH₄⁺, but NH₄⁺V_f was significantly higher with increased acetate. Thus, labile DOC V_f was not limited by the availability of NH₄⁺, but NH₄⁺V_f was limited by the availability of labile DOC. In contrast, neither glycine nor NH₄⁺V_f changed when released simultaneously indicating either that overall N‐uptake was saturated or that glycine and NH₄⁺ uptake were controlled by different factors. 4. Our results suggest that labile DOC and DON uptake can be equivalent to, or even higher than NH₄⁺ uptake, a solute known to be highly bioreactive, but unlike NH₄⁺ uptake, may not differ among land‐use types and seasons. Moreover, downstream export of nitrogen may be exacerbated by limitation of NH₄⁺ uptake by the availability of labile DOC in headwater streams from the agricultural Midwestern United States. Further research is needed to identify the factors that influence cycling of DOC and DON in streams.     

Continuous thermophilic composting (CTC) for rapid biodegradation and maturation of organic municipal solid waste

Fewer and fewer municipal solid wastes are treated by composting in China because of the disadvantages of enormous investment, long processing cycle and unstable products in a conventional composting treatment. In this study, a continuous thermophilic composting (CTC) method, only a thermophilic phase within the process, has been applied to four bench-scale composting runs, and further compared with a conventional composting run by assessing the indexes of pH, total organic carbon (TOC), total Kjeldahl nitrogen (TKN), C/N ratio, germination index (GI), specific oxygen uptake rate (SOUR), dissolved organic carbon (DOC) and dehydrogenase activity. After composting for 14 days, 16 days, 18 days and 19 days in the four CTC runs, respectively, mature compost products were obtained, with quality similar to or better than which had been stabilized for 28 days in run A. The products from the CTC runs also showed favorable stability in room temperature environment after the short-term composting at high temperature. The study suggested CTC as a novel method for rapid degradation and maturation of organic municipal solid wastes.     

Analysis of noise and bias in fermentation oxygen uptake rate data

The calculation of many derived fermentation variables such as the respiratory quotient (RQ) and mass transfer coefficient (K(L)a) requires the differences between the molar percentages of oxygen and carbon dioxide in the fermentor inlet and exit gas, called the %OUR and %CER. Noise and bias in %CER data is of order that in the exit gas carbon dioxide analysis. However, the relative amount of noise in the %OUR is one to two orders of magnitude greater than the noise in the raw oxygen analyses because the %OUR is calculated as a small difference between two large quantities. The noise in the %OUR is white with a Gaussian amplitude probability distribution of absolute standard deviation 0.0145. A chi-square filter of the %OUR data is shown to considerably improve the quality of the calculated RQ and K(L)a for a fermentation of Streptomyces clavuligerus. (c) 1992 John Wiley & Sons, Inc.     

On-line oxygen uptake rate and culture viability measurement of animal cell culture using microplates with integrated oxygen sensors

O2 uptake rates of animal cells (Chinese hamster ovary-CHO) were measured in 96-well microtiter plates by integrating with fluorescent sensors thereby measuring fluorescence intensity ratios of an O2-sensitive and an insensitive fluorophor. O2 consumption rate was estimated from measured dissolved O2 and from O2 mass transfer coefficient determined in advance. Specific uptake decreased with time from 3.2 x 10(-13) mol O2 cell(-1) h(-1) at 15 h cultivation to 1.8 x 10(-13) mol O2 cell(-1) h(-1) at 48 h. Specific O2 uptake was also determined by sampling from a spinner-flask culture giving identical values. A cell viability assay for cultures based on O2 measurements is described in which cells are incubated outside the fluorescence reader and then the dissolved O2 is measured only once at a fixed time after the start of incubation. This protocol can be directly applied for high-throughput measurements.     

Chemical and structural evolution of humic acids during organic waste composting

The effects of the composting process on the chemical and structural properties of humic acids have been studied in seven different organic waste mixtures from different origin. Only slight changes in elemental composition have been found in the humic acids after the composting process pointing to a more aromatic structure with higher proportions of oxygen and nitrogen. Functional groups were the most sensitive to the changes caused by the composting process showing a marked increase in the total acidity and phenolic, carboxyl and carbonyl groups. Gel permeation chromatography showed a slight increase in the average molecular weight of the humic acids. Infrared spectroscopy did not show important differences in humic acid structure but a clear decrease in the intensity of the bands in the region 3000–2850 cm^-1 corresponding to the aliphatic fractions. As a general result, the composting process yields humic acids in which the elemental and functional composition are closer to that of the more humified soil humic acids.     

The effect of dissolved oxygen and aeration rate on antibiotic production of Streptomyces fradiae

Different dissolved oxygen concentrations and aeration rates were imposed on a stable mutant of Streptomyces fradiae during the antibiotic-producing phase. At high aeration rate (1 vvm), the tylosin yield in the fermentor broth with dissolved oxygen (DO) concentrations controlled close to 100% saturation (6-8 ppm) increased 10% as against uncontrolled. The rates of cellular growth, oil consumption, and tylosin production were severely reduced when DO concentration fell below 25% saturation, but all resumed to their initial rates when DO was raised to saturation level again. The DO concentration in combination with air flow rate affected the pattern of the antibiotics produced. At high DO levels, an additional macrolide antibiotic, macrocin, was synthesized to more than one-third the amount of tylosin at high aeration rate (1 vvm). On the other hand, tylosin production rate remained constant and no significant amount of macrocin was produced at low aeration rate (0.2 vvm).     

Colloidal and dissolved organic matter in lake water: Carbohydrate and amino acid composition,and ability to support bacterial growth

Bacterial utilization of dissolved organic matter (DOM) was studied in water from a humic and a clearwater oligotrophic lake. Indigenous bacteria were inoculated into either 0.2 m natural filtered lake water, or lake water enriched fivefold with colloidal DOM >100 kD but below 0.2 m. Consumption of DOM was followed from changes in concentrations of total dissolved organic carbon (DOC), dissolved combined and free carbohydrates and amino acids (DCCHO and DFCHO, and DCAA and DFAA, respectively) and by uptake of monosaccharide and amino acid radioisotopes. DCCHO and DCAA made up 8% (humic lake) to 33–44% (clear-water lake) of the natural DOC pools, while DFCHO and DFAA contributed at most 1.7% to the DOC pools. Addition of >100 kD DOM increased the DOC concentrations by 50% (clearwater lake) to 92% (humic lake), but it only resulted in a higher bacterial production (by 63%) in the humic lake. During the incubations 13 to 37% of the DOC was assimilated by the bacteria, at estimated growth efficiencies of 4–8%. Despite the measured reduction of DOC, statistically significant changes of specific organic compounds, especially of DCCHO and DCAA, generally did not occur. Probably the presence of high molecular weight DOC interfered with the applied analytical procedures. Addition of radiotracers indicated, however, that DFAA sustained 17–58% and 29–100% of the bacterial carbon and nitrogen requirements, respectively, and that glucose met 1–3% of the bacterial carbon requirements. Thus, our experiments indicate that radiotracers, rather than measurements of concentration changes, should be used in studies of bacterial utilization of DOC in freshwaters with a high content of humic or high molecular weight organic matter.     

Effects of salinity and light on organic carbon and nitrogen uptake in a hypersaline microbial mat

Utilization of dissolved organic matter (DOM) is thought to be the purview of heterotrophic microorganisms, but photoautotrophs can take up dissolved organic nitrogen (DON) and dissolved organic carbon (DOC). This study investigated DOC and DON uptake in a laminated cyanobacterial mat community from hypersaline Salt Pond (San Salvador, Bahamas). The total community uptake of (3)H-labeled substrates was measured in the light and in the dark and under conditions of high and low salinity. Salinity was the primary control of DOM uptake, with increased uptake occurring under low-salinity, 'freshened' conditions. DOC uptake was also enhanced in the light as compared with the dark and in samples incubated with the photosystem II inhibitor 3(3,4-dichlorophenyl)-1, 1-dimethylurea, suggesting a positive association between photosynthetic activity and DOC uptake. Microautoradiography revealed that some DOM uptake was attributed to cyanobacteria. Cyanobacteria DOM uptake was negatively correlated with that of smaller filamentous microorganisms, and DOM uptake by individual coccoid cells was negatively correlated with uptake by colonial coccoids. These patterns of activity suggest that Salt Pond microorganisms are engaged in resource partitioning, and DOM utilization may provide a metabolic boost to both heterotrophs and photoautrophs during periods of lowered salinity.     

On-line gas analysis in animal cell cultivation: II. Methods for oxygen uptake rate estimation and its application to controlled feeding of glutamine

Different methods for oxygen uptake rate (OUR) determinations in animal cell cultivation were investigated using a high quality mass spectrometer. Dynamic measurements have considerable disadvantages because of disturbances of the growing cells by the necessary variations of dissolved oxygen concentration. Only infrequent discrete measurements are possible using this method. Stationary liquid phase balance yielded better results with much higher frequency. Gas phase balancing has the advantage of not requiring dissolved oxygen measurement and knowledge of K(L)a, both of them are easily biased. It was found that simple gas phase balancing is either very inaccurate (error larger than expected signal) or very slow, with gas phase residence times of several hours. Therefore, a new method of aeration was designed. Oxygen and CO(2) transfer are mainly achieved via sparging. The gas released to the headspace is diluted with a roughly 100-fold stream of an inert gas (helium). Through this dilution, gas ratios are not changed for O(2), CO(2), Ar, and N(2). The measurement of lower concentrations (parts per million and below) is easy using mass spectrometry with a secondary electron multiplier. With this new method an excellent accuracy and sufficient speed of analysis were obtained. All these on-line methods for OUR measurement were tested during the cultivation of animal cells. The new method allowed better study of the kinetics of animal cell cultures as was shown with a hybridoma cell line (HFN 7.1, ATCC CRL 1606) producing monoclonal antibodies against human fibronectin. With the aid of these methods it was possible to find a correlation between a rapid decrease in oxygen uptake rate (OUR) and glutamine concentration. The sudden decrease in OUR can be attributed to glutamine depletion. This provided a basis for the controlled addition of glutamine to reduce the formation of ammonia produced by hydrolysis. This control method based on OUR measurement resulted in increased cell concentration and threefold higher product concentration. (c) 1995 John Wiley & Sons, Inc.     

Relationship between oxygen uptake rate and time of infection of Sf9 insect cells infected with a recombinant baculovirus

Oxygen uptake rates (OUR) of Sf9 insect cells propagated in a serum-free medium (SF900II, Gibco) and of cells infected with a recombinant AcNPV were investigated before and after infection in a laboratory-scale bioreactor. The volumetric OURs of uninfected and exponentially growing cells were found to be proportional to the cell density. For infected cultures, the specific OUR of cells increased immediately after addition of virus and a maximum of 1.3 times the value of uninfected cells was noted for all the cultures between 8 to 30 hours post infection, which coincides with the period at which most viral replication and the majority of DNA synthesis takes place. It was observed that the rate of rise in the specific OUR decreased as the cell density at the time of infection increased, which meant that the later the infection, the later the maximum sOUR was observed. We therefore suggest that OUR measurement can be used to reflect the efficiency of a batch infection. Carbohydrate and amino acid consumption rates from an infected run were analysed in an effort to identify substrate(s) that may be used at increased rates to fuel the rise in oxygen demand observed early in the infection cycle. No observable rise in the consumption rates of glucose or glutamine, which are the major energy sources for animal cells, were seen after infection but an increase in the consumption rates of some amino acids suggests that infected Sf9 cells may utilise amino acids at an enhanced rate for energy post infection.     

Specific oxygen, ammonia, and nitrate uptake rates of a biological nutrient removal process treating elevated salinity wastewater

Anaerobic/anoxic/aerobic systems inoculated without and with NaCl acclimated cultures, i.e., Models A and B, respectively, were fed with a synthetic wastewater at various salinity levels. After achieving a steady state, the systems were shocked with 70 g/l NaCl for four consecutive days before returning to pre-shock conditions. At the steady-state, the specific oxygen uptake rates (SOURs) increased with an increase of sodium chloride concentration (from 5.40 to 9.72 mg O₂/g mixed liquor suspended solids (MLSS)-h at 0–30 g/l NaCl for Model A and from 6.84 to 17.64 mg O₂/g MLSS-h at 5–30 g/l NaCl for Model B). In contrast, the specific ammonia uptake rate (SAUR) and specific nitrate uptake rate (SNUR) decreased with increasing chloride concentration (from 4.76 to 2.14 mg NH₃–N/g MLSS-h and 2.50 to 1.22 NO3–N/g MLSS-h, for Model A, and from 3.84 to 2.71 mg NH₃–N/g MLSS-hr and 2.54 to 1.82 mg NO₃–N/g MLSS-hr, for Model B). During the shocked period, the SOUR in most scenarios increased whereas the SAUR and SNUR tended to decrease. The impact of the chloride shock on nitrifiers was more obvious than on denitrifiers; however, after a certain recovery period, the activities of both nitrifiers and denitrifiers in terms of SAUR and SNUR were approximately the same as those prior to shock.     

On-line gas analysis in animal cell cultivation: I. Control of dissolved oxygen and pH

To monitor gas reaction rates in animal cell culture at constant dissolved oxygen concentration (DO) and constant pH it was necessary to develop improved control methods. Decoupling of both controllrs was obtained by manipulation of molar fractions of oxygen and carbon dioxide in the gas phase. Two pairs of DO and pH controllers were designed and tested both in simulation and exprimental runs. The first controller pair was developed for headspace aeration only, whereas the second controller pair was designed for bubble aeration using a microsparger and flushing the headspace with helium. pH was controlled by a conventional discrete PID controller in its velocity form. For DO control two linear state space feedback controllers with parameter adaptation were established. In these controllers the oxygen uptake rate (OUR) was considered as a disturbance and was not included in the mathematical model. The feedback gain adaptation was based on the difference between the actual molar fraction of oxygen at time step n and the initial molar fraction. This difference is related to OUR and was used to increase or decrease the state feedback controller gain (k and k(1), respectively) in a slow manner. With these controllers it was possible to get an excellent online estimate of OUR. In the case of bubble aeration a simple gas phase mass balance was sufficient, whereas during the headspace aeration a liquid phase balance was required. It has been shown that determination of OUR using gas balance requires a significantly better controller performance compared to just keeping DO and pH within reasonable limits. (c) 1995 John Wiley & Sons, Inc.     

NIR spectroscopy,mineral nitrogen analysis and soil incubations for the prediction of crop uptake of nitrogen during the growing season

To predict the amount of N taken up in above-ground plant parts during the growing season, initial mineral soil N, a soil incubation method, soil organic matter and NIR data were compared as predictors. Soil samples were taken from 15 plots cropped with winter wheat on a farm in south-western Sweden. The plots were not fertilized with N during the 1997 growing season. N contents in above-ground plant parts were measured in mid-June and in mid-August. All methods were capable of predicting the crop uptake of N reasonably well. NIR data gave at least as good predictions as the best traditional method, initial soil NO₃-N. The most important wavelengths, around 1400 and 1700 nm, and above 2000 nm, coincide with the wavelengths reported earlier to be important for the prediction of soil organic matter. However, the data suggest that other soil components influencing mineralization are also spectrally active. Since very few samples were taken, the studies need to be extended in order to be able to use the method in practice. It is recommended that further studies be instigated for the possibility of using the same NIR calibration over several years and to clarify the spatial regions that the calibrations can cover. This revised version was published online in June 2006 with corrections to the Cover Date.     

A simple model for estimating the contribution of nitrogen mineralization to the nitrogen supply of crops from a stabilized pool of soil organic matter and recent organic input

A simple model was developed to estimate the contribution of nitrogen (N) mineralization to the N supply of crops. In this model the soil organic matter is divided into active and passive pools. Annual soil mineralization of N is derived from the active pool. The active pool comprises stabilized and labile soil organic N. The stabilized N is built up from accumulated inputs of fresh organic N during a crop rotation but the labile N is a fraction of total N added, which mineralizes faster than the stabilized N. The passive pool is considered to have no participation in the mineralization process. Mineralization rates of labile and stabilized soil organic N from different crop residues decomposing in soil were derived from the literature and were described by the first-order rate equation dN/dt =-K*N, where N is the mineralizable organic N from crop residues andK is a constant. The data were groupedK ₁ by short-term (0–1 year) andK ₂ by long-term (0–10 years) incubation. Because the range of variation inK ₂ was smaller than inK ₁ we felt justified in using an average value to derive N mineralization from the stabilized pool. The use of a constant rate ofK ₁ was avoided so net N mineralization during the first year after addition is derived directly from the labile N in the crop residues. The model was applied to four Chilean agro-ecosystems, using daily averages of soil temperature and moisture. The N losses by leaching were also calculated. The N mineralization varied between 30 and 130 kg N ha^–1 yr^–1 depending on organic N inputs. Nitrogen losses by leaching in a poorly structured soil were estimated to be about 10% of total N mineralized. The model could explain the large differences in N- mineralization as measured by the potential N mineralization at the four sites studied. However, when grassland was present in the crop rotation, the model underestimated the results obtained from potential mineralization.     

Correlation of O2 uptake rate and mitochondrial activity in carrot cell cultures exposed to laminar fluid shear

Measuring uncoupled oxygen uptake rate (OUR) could provide a convenient method for quantifying changes in the metabolic activity of plant cultures caused by hydrodynamic shear. Experiments on Daucus carota (carrot) cells were performed in a novel O₂-permeable Couette viscometer at varying levels of laminar shear (6 to 100 N m^–2). When the uncoupled OUR of the cells was compared with mitochondrial activity (determined by 2,3,5-triphenyl tetrazolium chloride assay), a significant correlation was observed (R=0.91 by linear regression).     

Anaerobic contribution to the time to exhaustion at the minimal exercise intensity at which maximal oxygen uptake occurs in elite cyclists, kayakists and swimmers

Using 23 elite male athletes (8 cyclists, 7 kayakists, and 8 swimmers), the contribution of the anaerobic energy system to the time to exhaustion (t _lim) at the minimal exercise intensity (speed or power) at which maximal oxygen uptake (V˙O_{2 max}) occurs (I _{V˙O2 max}) was assessed by analysing the relationship between the t _lim and the accumulated oxygen deficit (AOD). After 10-min warming up at 60% of V˙O_{2 max}, the exercise intensity was increased so that each subject reached his I _V˙O2max in 30 s and then continued at that level until he was exhausted. Pre-tests included a continuous incremental test with 2 min steps for determining the I _V˙O2max and a series of 5-min submaximal intensities to collect the data that would allow the estimation of the energy expenditure at I _V˙O2max . The AOD for the t _lim exercise was calculated as the difference between the above estimation and the accumulated oxygen uptake. The mean percentage value of energy expenditure covered by anaerobic metabolism was 15.2 [(SD 6)%, range 8.9–24.1] with significant differences between swimmers and kayakists (16.8% vs 11.5%, P≤0.05) and cyclists and kayakists (16.4% vs 11.5%, P≤0.05). Absolute AOD values ranged from 26.4 ml · kg⁻¹ to 83.6 ml · kg⁻¹ with a mean value of 45.9 (SD 18) ml · kg⁻¹. Considering all the subjects, the t _lim was found to have a positive and significant correlation with AOD (r = 0.62, P≤0.05), and a negative and significant correlation with V˙O_{2 max} (r = −0.46, P≤0.05). The data would suggest that the contribution of anaerobic processes during exercise performed at I _V˙O2max should not be ignored when t _lim is used as a supplementary parameter to evaluate specific adaptation of athletes. Accepted: 17 December 1996     

Effects of different molecular weight fractions of dissolved organic matter on the growth of bacteria,algae and protozoa from a highly humic lake

Effects of different molecular size fractions (< 1000 MW, < 10 000 MW, < 100 000 MW and <0.1 μm) of dissolved organic matter (DOM) on the growth of bacteria, algae and protozoa from a highly humic lake were investigated. DOM from catchment drainage water as well as from the lake consisted mostly (59–63%) of high molecular weight (HMW) compounds (> 10 000 MW). With excess inorganic nutrients, the growth rate and yield of bacteria were almost identical in all size fractions. However, in < 1000 MW fractions and with glucose added, a longer lag phase occurred. Without added nutrients both the growth rates and biomasses of bacteria decreased towards the smaller size fractions and the percentage of dissolved organic carbon (DOC) used during the experiment and the growth efficiency of bacteria were lower than with excess nutrients. The growth efficiency of bacteria was estimated to vary between 3–66% in different MW fractions, largely depending on the nutrient concentrations, but the highest growth efficiencies were observed in HMW fractions and with glucose. The growth of algae was clearly lowest in the < 1000 MW fraction. In dim light no net growth of algae could be found. In contrast, added nutrients substantially enhanced algal growth and in deionized water with glucose, algae achieved almost the same growth rate and biomass as in higher MW fractions of DOM. The results suggested that bacteria and some algae were favoured by DOM, but protozoans seemed to benefit only indirectly, through bacterial grazing. The utilization of DOM by bacteria and algae was strongly affected by the availability of phosphorus and nitrogen.