Effect of iron on growth and lipid accumulation in Chlorella vulgaris

The economic feasibility of algal mass culture for biodiesel production is enhanced by the increase in biomass productivity and storage lipids. Effect of iron on growth and lipid accumulation in marine microalgae Chlorella vulgaris were investigated. In experiment I, supplementing the growth media with chelated FeCl3 in the late growth phase increased the final cell density but did not induce lipid accumulation in cells. In experiment II, cells in the late-exponential growth phase were collected by centrifugation and re-inoculated into new media supplemented with five levels of Fe3+ concentration. Total lipid content in cultures supplemented with 1.2 x 10(-5) mol L(-1) FeCl3 was up to 56.6% biomass by dry weight and was 3-7-fold that in other media supplemented with lower iron concentration. Moreover, a simple and rapid method determining the lipid accumulation in C. vulgaris with spectrofluorimetry was developed.     

Nicotinate catabolism is dispensable and nicotinate anabolism is crucial in Azorhizobium caulinodans growing in batch culture and chemostat culture on N2 as The N source.

When Azorhizobium caulinodans was grown in chemostat cultures with N2 as the N source at a constant dilution rate of 0.1 h-1 in media with a constant concentration (50 mM) of succinate and variable concentrations (1.5 to 585 microM) of nicotinate, neither the growth yield on succinate, the specific rate of O2 consumption, nor the specific rate of CO2 production showed linear regression with the concentration of nicotinate. Moreover, for transient continuous cultures in which the nicotinate concentration was gradually lowered, growth parameters remained unchanged until an apparently critical level of 0.7 microM nicotinate was reached. Below this nicotinate level, an immediate washout of the chemostat population began. A. caulinodans nicotinate hydroxylase-negative mutant 61007, unable to catabolize nicotinate, and the wild type behaved similarly. Thus, for continuous cultures supplied with N2 as the N source, submicromolar concentrations of nicotinate both sustained pyridine nucleotide biosynthesis at sufficient levels and precluded the use of nicotinate as a catabolic substrate. Furthermore, when more nicotinate was provided, dual succinate-nicotinate limitation in continuous cultures did not occur. Finally, when nicotinate is present in suboptimal concentrations, the specific growth rate is directly proportional to the amount of nicotinate present per unit of biomass. By contrast, in batch cultures with different nicotinate concentrations and with either succinate or lactate as the carbon and energy source, anomalous growth curves were obtained. With a low concentration (1.5 microM) of nicotinate, growth on N2 occurred, albeit at low rates. With a high concentration (195 microM) of nicotinate, growth on N2 was temporarily stimulated, but nicotinate was quickly exhausted and growth was thereafter nicotinate limited. Continuous supplementation of batch cultures with nicotinate allowed only transient exponential growth followed by linear growth. Thus, also for batch cultures, nicotinate catabolism is dispensable, although a high concentration of nicotinate temporarily stimulates growth on N2. Ut us concluded that A. caulinodans is a true diazotroph.     

The relation between nutrient solution concentration and growth and ion absorption of peas (Pisum sativum L.)

Summary Studies have been made on the growth of peas in Richter's nutrient solution at concentrations of 1.0 (original solution), 0.1, 0.01, and 0.001; instead of employing flowing cultures, the solutions were vigorously stirred and continually replaced by fresh solution to maintain the concentration within close limits (±5%). Maximum growth occurred at the 1 and 0.1 concentrations; at lower concentrations growth was significantly depressed, but no deficiency symptoms were observed. The relative growth rate decreased regularly with decreasing concentration. No correspondence was observed between the reduction in solution concentration and that in the yield; for a concentration decrease of 1000 times, dry matter production was depressed only 4.85 times. With decreasing solution concentration the chlorophyll content however was depressed more than growth and at the lowest concentration was less than 1/80th of that in the original solution. As a result the net assimilation rate, calculated on the basis of dry matter production per unit chlorophyll content, increased sharply with decreasing nutrient solution concentration. Even at the lowest concentration the plants continued to grow throughout the course of the experiment.     

Studies on production of ajmalicine in shake flasks by multiple shoot cultures of Catharanthus roseus

The effects of different concentrations of indole-3-acetic acid (IAA) and benzyladenine (BA) on production of ajmalicine by multiple shoot cultures of Catharanthus roseus (C. roseus) were studied. By supplementing Murashige and Skoog's (MS) medium with a high concentration of IAA (11.42 microM) and a low concentration of BA (2.22 microM), shoot cultures accumulated high levels of ajmalicine. When culture medium was fortified with a low concentration of IAA (2.85 microM) and a high concentration of BA (8.90 microM), shoots released high levels of ajmalicine into the culture medium. Quantification of ajmalicine was performed by high performance liquid chromatography (HPLC). The highest concentration of ajmalicine production (0.166% dry wt) was obtained by shoot cultures grown in MS medium containing IAA (11.42 microM) on 20 days of cultivation. Shoot cultures accumulated ajmalicine 4.2-fold more in IAA (11.42 microM) supplemented medium compared with the high concentration of BA (8.90 microM). The content of ajmalicine concentration in the medium was quantified. Shoot cultures grown in BA (8.90 microM) supplemented medium released the maximum production of ajmalicine (0.853 g/L) into the culture medium after 15 days of cultivation. The experimental data show that the secretion of ajmalicine was 2-fold more into the culture medium supplemented with a high concentration of BA compared to that with a low concentration of BA. Data presented here show that production of ajmalicine by shoot cultures is not correlated with growth rate. Dimeric indole alkaloids vincristine and vinblastine were not present in shoot cultures. Ajmalicine production by shoot cultures was 2.4-fold higher compared to leaves of 1-year-old naturally grown plants.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Effect of iron ions on the antioxidant enzyme activities in yeast Saccharomyces cerevisiae

A complex of physiological and biochemical indices has been compared in wild and isogenic catalase-deficient strains of Saccharomyces cerevisiae grown on the media with different iron ion concentrations is 2 times higher in cytosolic catalase deficient yeast. Superoxide dismutase activity grown in the medium with 500 microM of ferrous sulphate. Under such conditions, peroxisomal catalase deficient yeast had a 2-fold decreased activity of superoxide dismutase. There is a significant difference between TBA-reactive substances content of the wild and cytosolic catalase deficient strain. It has been suggested that the repletion of iron ions in the growth medium leads to the formation of lipid oxidation products. Catalase prevents TBA-reactive substances formation in the given conditions and plays a protective role.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa.

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Effect of Helium Gas at Elevated Pressure on Iron Transport and Growth of Escherichia coli

Helium at an ambient pressure of 68 at m with 0.2 atm of O(2) shortened by 1 to 1.5 h the lag phase for growth of Escherichia coli in minimal medium supplemented with 2 muliters of cell-free culture filtrate (CFF) per ml or with 1 muM 2,3-dihydroxybenzoylserine (DHBS), an iron chelator. The lag phase of cultures not exposed to helium could be shortened by use of supplements, but higher concentrations were required-10 to 30 muliters of CFF per ml or 10 to 50 muM DHBS. Strain AN 193 of E. coli, which requires the DHBS precursor 2,3-dihydroxybenzoic acid (DHBA), grew well in media with 10 muM DHBA when exposed to helium at 68 atm, whereas 100 muM DHBA was required for growth in unexposed cultures. In the presence of 100 muM DHBA plus 1.0 muM ethylenediaminetetraactic acid, growth was inhibited at 1 and 68 atm. Growth was restored, however, by the addition of 0.1 muM FeSO(4) at 68 atm and 1.0 muM FeSO(4) at 1 atm, but lag times were invariably shorter in the pressurized cultures. Hydrostatic pressures of 68 atm did not reduce the lag phase in the presence of CFF, DHBS, or DHBA. Our results suggest that 68 atm of helium pressure, but not hydrostatic pressure, elicited a more rapid transport of iron into the cells.     

Growth and hemolysin production by Haemophilus pleuropneumoniae cultivated in a chemically defined medium

A chemically defined medium (CDM) has been developed which supports both growth and hemolysin production by Haemophilus pleuropneumoniae. Although the growth rate in stationary cultures was substantially slower in CDM than in trypticase soy broth plus 0.6% yeast extract (TSBYE) and slightly slower than in heart infusion broth (HIB), extracellular hemolysin activity in CDM was slightly higher than in HIB and 16-fold greater than in TSBYE. Maximum hemolytic activity was produced in CDM in early to mid log phase of growth. Hemolytic activity in sterile, cell-free culture supernatant fluids persisted for over 10 days at 4 degrees C and 3-5 days at 37 degrees C, but was completely destroyed at 56 degrees C after 30 min. Total hemolysin inactivation was also achieved in the presence of trypsin or pronase (10 units/mL), but no decrease in hemolytic activity was noted in the presence of DNase or RNase. Iron had little effect on the hemolytic activity in the early stages of growth. However, in the later stages of growth, iron had a pronounced effect with hemolytic activity decreasing as the iron concentration increased from 1 to 500 microM. None of these iron concentrations had any effect on the hemolytic activity when added directly to prepared cell-free culture supernatant fluids. The extracellular hemolysin produced by H. pleuropneumoniae in CDM appears to be a heat-labile protein the activity of which is influenced by iron at certain phases of growth.     

Effect of cadmium on the growth of Chlorella vulgaris and Stichococcus bacillaris

The growth of Chlorella vulgaris and Stichococcus bacillaris cultures in media containing from 20 to 100 mg Cd/l was studied. The examined strains were found to be highly resistant to the action of cadmium since the highest concentration of the metal used limited the production of dry weight (during 5 days of cultivation) by less than 50%. The lower production of chlorophyll a by S. bacillaris cultures in media containing from 60 to 100 mg Cd/l and 2-fold elongation of the cells point to lower tolerance of the strain to cadmium than that shown by C. vulgaris.     

Ultrafiltration to remove endotoxins and other cytokine-inducing materials from tissue culture media and parenteral fluids.

The presence of small amounts of endotoxins are often undesirable when investigating cytokines such as interleukin-1 and tumor necrosis factor alpha. Polymyxin B, widely used to block endotoxins, does not block several forms of endotoxins, and at high concentrations, polymyxin B itself stimulates interleukin-1 production. Human peripheral blood mononuclear cells are highly sensitive to endotoxins; they respond with cytokine production to endotoxins at concentrations of 10-50 pg/ml and detect pyrogenic materials nonreactive in the Limulus test. In the present study, ultrafiltration using polysulfone filters was found to remove all interleukin-1- and tumor necrosis factor alpha-inducing substances produced in E. coli cultures. Interleukin-1- and tumor necrosis factor alpha-inducing substances derived from Pseudomonas aeruginosa cultures were also rejected by the filters. Ultrafiltration is therefore a convenient and effective procedure to remove interleukin-1 and tumor necrosis factor alpha-inducing substances from parenteral fluids and solutions that come in contact with blood such as fluids used in hemodialysis. This technique is also applicable for the large-scale production of culture media for mammalian cell expression of recombinant, pyrogen-free proteins intended for use in humans.     

Hydrogen cyanide production by Pseudomonas aeruginosa at reduced oxygen levels

Hydrogen cyanide production by Pseudomonas aeruginosa growing in a synthetic medium required aerobosis but operated efficiently at low dissolved oxygen concentration. Half maximum levels of cyanogenesis occurred at 0.015 microM oxygen; maximum cyanogenesis occurred over a wide range, 0.1-180 microM, of oxygen concentrations. These cells lost the ability to produce cyanide upon aerobic incubation in the absence of both the carbon energy source (L-glutamate) and the metabolic precursor of hydrogen cyanide (glycine). This loss of cyanogenesis was dependent on oxygen concentration; 1.0 microM oxygen produced no detectable loss, whereas 180 microM oxygen caused a rapid decline in cyanogenic ability. The endogenous cyanide production rate of cells in the presence of carbon energy source was not significantly influenced by oxygen concentration. During the batch culture cycle, the acquisition of the ability to produce HCN was preceded by oxygen reduction to growth-limiting levels. Cells which had lost the ability to produce hydrogen cyanide by oxygen treatment required protein synthesis before they could again become cyanogenic.     

Direct and indirect bacterial killing functions of neutrophil defensins in lung explants

Studies of the antimicrobial activity of neutrophil defensins have mostly been carried out in microbiological media, and their effects on the host defense in physiological conditions are unclear. We examined 1) the antibacterial activity of defensins in physiological media with and without lung tissue present, 2) the effect of defensins on hydrogen peroxide (H(2)O(2)) production by lung tissue that had been exposed to bacteria, and 3) the effect of diphenyleneiodonium (DPI), an inhibitor of reactive oxygen species formation, on the antibacterial activity of defensins in the presence of lung tissue. Defensins were incubated with Escherichia coli or Pseudomonas aeruginosa in the absence or presence of primary cultured mouse lung explants. Defensins reduced bacterial counts by approximately 65-fold and approximately 25-fold, respectively, at 48 h; bacterial counts were further decreased by approximately 600-fold and approximately 12,000-fold, respectively, in the presence of lung tissue. Defensins induced H(2)O(2) production by lung tissue, and the rate of killing of E. coli by defensins was reduced by approximately 2,500-fold in the presence of 10 microM DPI. We conclude that defensins exert a significant antimicrobial effect under physiological conditions and that this effect is enhanced in the presence of lung tissue by a mechanism that involves the production of reactive oxygen species.     

Inhibition of cholesterol synthesis and cell growth by 24(R,S),25-iminolanosterol and triparanol in cultured rat hepatoma cells

24(R,S),25-Iminolanosterol (IL) and triparanol added to cultures of rat hepatoma cells, H4-II-C3 (H4), interrupt the conversion of lanosterol to cholesterol and, depending on their concentrations, cause the accumulation in the cells of intermediates in the lanosterol to cholesterol conversion. At 45 microM, both substances cause the accumulation of 5 alpha-cholesta-8(9),24-dien-3 beta-ol (zymosterol), and at the low concentration of 4.5 microM, they cause the accumulation of cholesta-5.24-dien-3 beta-ol (desmosterol). The effect of intermediate concentrations of 9 or 22.5 microM of either substance is to cause the accumulation in the cells of three sterols: cholesta-5,7,24-trien-3 beta-ol, zymosterol, and desmosterol. The synthesis of these intermediary sterols, not found normally in H4 cells, is particularly pronounced in cultures kept in lipid-depleted media that contain the inhibitors and proceeds by the use of endogenous substrates at the expense of cholesterol. The synthesis of cholesterol from [14C]acetate or [2-14C]mevalonate is completely blocked by either inhibitor even at 4.5 microM. IL or triparanol inhibits the growth of H4 cells. Cells seeded into either full growth or lipid-depleted medium containing 22.5 microM IL will not grow unless the media are supplemented with low density lipoproteins (60 micrograms/ml). Supplementation of the media with 4.6 mM mevalonate does not counteract the inhibitory effect of IL on cell growth.     

Enterobactin-mediated iron transport in Pseudomonas aeruginosa.

A pyoverdine-deficient strain of Pseudomonas aeruginosa was unable to grow in an iron-deficient minimal medium in the presence of the nonmetabolizable iron chelator ethylene diamine-di(omega-hydroxyphenol acetic acid) (EDDHA), although addition of enterobactin to EDDHA-containing minimal media did restore growth of the pyoverdine-deficient P. aeruginosa. Consistent with the apparent ability of enterobactin to provide iron to P. aeruginosa, enterobactin-dependent 55Fe3+ uptake was observed in cells of P. aeruginosa previously grown in an iron-deficient medium containing enterobactin (or enterobactin-containing Escherichia coli culture supernatant). This uptake was energy dependent, was observable at low concentrations (60 nM) of FeCl3, and was absent in cells cultured without enterobactin. A novel protein with a molecular weight of approximately 80,000 was identified in the outer membranes of cells grown in iron-deficient minimal medium containing enterobactin, concomitant with the induction of enterobactin-dependent iron uptake. A Tn501 insertion mutant lacking this protein was isolated and shown to be deficient in enterobactin-mediated iron transport at 60 nM FeCl3, although it still exhibited enterobactin-dependent growth in iron-deficient medium containing EDDHA. It was subsequently observed that the mutant was, however, capable of enterobactin-mediated iron transport at much higher concentrations (600 nM) of FeCl3. Indeed, enterobactin-dependent iron uptake at this concentration of iron was observed in both the mutant and parent strains irrespective of whether they had been cultured in the presence of enterobactin. Apparently, at least two uptake systems for ferrienterobactin exist in P. aeruginosa: one of higher affinity which is specifically inducible by enterobactin under iron-limiting conditions and the second, of lower affinity, which is also inducible under iron-limiting conditions but is independent of enterobactin for induction.     

IRON-MEDIATED CHANGES IN THE GROWTH OF LAKE ERIE PHYTOPLANKTON AND AXENIC ALGAL CULTURES1

The effect of iron enrichment on algal growth and photosynthesis was investigated using natural assemblages of Lake Erie phytoplankton and axenic cultures of Anabaena, Scenedesmus and Selenastrum. Cell yield and photosynthesis were frequently inhibited in the presence of unchelated iron over the range of 3.6 to 53.7 μM iron as FeCl₃. In lake water and in a defined medium with low nutrient concentrations, the degree of inhibition by iron could be reduced by chelating the iron with EDTA or by enriching the cultures with phosphorus. Chemical analyses revealed that the EDTA efectively reduced the ability of the ferric iron to remove soluble phosphorus from the media. EDTA was also observed to reduce rather than enhance iron uptake by axenic cultures of A. flos-aquae. These data support the hypothesis that additions of EDTA to low-nutrient media may serve to stimulate algal growth in the presence of iron by preventing the iron from altering extracellular concentrations of soluble ions essential for algal metabolism. In medium with high nutrient concentrations, the soluble phosphorus concentration was not appreciably altered by either EDTA-chelated or unchelated iron enrichment (0.9 to 53.7 μM). Instead, the observed enhancement of cell yield by EDTA-chelated iron in nutrient-rich media appeared to be due to the direct effect of iron on intracellular metabolic processes.     

THE ANTIBACTERIAL SUBSTANCES OF THE MARINE ALGA STICHOCHRYSIS IMMOBILIS (CHRYSOPHYTA)12

Two substances with antibacterial activity have been concentrated from the culture medium of Stichochrysis immobilis before senescence occurred, and purified, by a Sephadex G-25 filtration followed by 2-dimensional thin-layer chromatography on Merck F-254 silica gel plates. One of the substances is obviously a peptide; 11 different amino acids are linked in a 30 amino acid molecule (MW 3400). It was not possible to assign a chemical group to the second substance (MW ? 2600); it may be related to “humic acids” (Kalle's gelbstoff). Growth of the most sensitive bacteria was completely inhibited by 20-fold, or higher, concentrations of substances from the algal culture filtrate (assuming no loss of substances during the concentration processes). The 54 strains of test bacteria were of various origins, though mostly isolated from algal cultures or obtained directly from the marine environment.     

Effect of iron concentration on toxin production in Campylobacter jejuni and Campylobacter coli

The effect of iron concentrations in culture media on supernatant yields of campylobacter cytotonic toxin (CCT) was studied. Of the 118 Campylobacter spp. strains surveyed, 78.8% produced toxin in brucella broth or in casamino acids--yeast extract (CYE) broth. When the iron concentration of CYE was increased from 0.44 microgram/mL (7.9 microM) to 0.65 microgram/mL (11.6 microM) by the addition of ferric chloride, 94.9% of the strains were positive for toxin in a ganglioside GM1 based, enzyme-linked immunosorbent assay, using antibody to affinity-purified CCT. The addition of iron as ferrous sulfate was less effective. When four toxin-positive strains were grown in a deferrated medium of conalbumin-treated CYE with 0.04-0.08 microgram iron/mL (0.72-1.43 microM), two of the culture supernatants became negative (absorbance at 410 nm, less than 0.1 and less than 10 ng CCT/mL), and two produced about 90% less CCT but were still classified as positive (absorbance, greater than or equal to 0.1 and greater than or equal to 10 ng CCT/mL). It was therefore concluded that the production of CCT by Campylobacter spp. is influenced by iron concentration.     

Heterotrophic bacterial growth efficiency and community structure at different natural organic carbon concentrations

Batch cultures of aquatic bacteria and dissolved organic matter were used to examine the impact of carbon source concentration on bacterial growth, biomass, growth efficiency, and community composition. An aged concentrate of dissolved organic matter from a humic lake was diluted with organic compound-free artificial lake water to obtain concentrations of dissolved organic carbon (DOC) ranging from 0.04 to 2.53 mM. The bacterial biomass produced in the cultures increased linearly with the DOC concentration, indicating that bacterial biomass production was limited by the supply of carbon. The bacterial growth rate in the exponential growth phase exhibited a hyperbolic response to the DOC concentration, suggesting that the maximum growth rate was constrained by the substrate concentration at low DOC concentrations. Likewise, the bacterial growth efficiency calculated from the production of biomass and CO(2) increased asymptotically from 0.4 to 10.4% with increasing DOC concentration. The compositions of the microbial communities that emerged in the cultures were assessed by separation of PCR-amplified 16S rRNA fragments by denaturing gradient gel electrophoresis. Nonmetric multidimensional scaling of the gel profiles showed that there was a gradual change in the community composition along the DOC gradient; members of the beta subclass of the class Proteobacteria and members of the Cytophaga-Flavobacterium group were well represented at all concentrations, whereas members of the alpha subclass of the Proteobacteria were found exclusively at the lowest carbon concentration. The shift in community composition along the DOC gradient was similar to the patterns of growth efficiency and growth rate. The results suggest that the bacterial growth efficiencies, the rates of bacterial growth, and the compositions of bacterial communities are not constrained by substrate concentrations in most natural waters, with the possible exception of the most oligotrophic environments.     

Synergy of Fresh and Accumulated Organic Matter to Bacterial Growth

The main goal of this research was to evaluate whether the mixture of fresh labile dissolved organic matter (DOM) and accumulated refractory DOM influences bacterial production, respiration, and growth efficiency (BGE) in aquatic ecosystems. Bacterial batch cultures were set up using DOM leached from aquatic macrophytes as the fresh DOM pool and DOM accumulated from a tropical humic lagoon. Two sets of experiments were performed and bacterial growth was followed in cultures composed of each carbon substrate (first experiment) and by carbon substrates combined (second experiment), with and without the addition of nitrogen and phosphorus. In both experiments, bacterial production, respiration, and BGE were always higher in cultures with N and P additions, indicating a consistent inorganic nutrient limitation. Bacterial production, respiration, and BGE were higher in cultures set up with leachate DOM than in cultures set up with humic DOM, indicating that the quality of the organic matter pool influenced the bacterial growth. Bacterial production and respiration were higher in the mixture of substrates (second experiment) than expected by bacterial production and respiration in single substrate cultures (first experiment). We suggest that the differences in the concentration of some compounds between DOM sources, the co-metabolism on carbon compound decomposition, and the higher diversity of molecules possibly support a greater bacterial diversity which might explain the higher bacterial growth observed. Finally, our results indicate that the mixture of fresh labile and accumulated refractory DOM that naturally occurs in aquatic ecosystems could accelerate the bacterial growth and bacterial DOM removal.