High organic loading influences the physical characteristics of aerobic sludge granules

Aims:  The effect of high organic loading rate (OLR) on the physical characteristics of aerobic granules was studied.
Methods and Results:  Two column-type sequential aerobic sludge blanket reactors were fed with either glucose or acetate as the main carbon source, and the OLR was gradually raised from 6 to 9, 12 and 15 kg chemical oxygen demand (COD) m⁻³ d⁻¹. Glucose-fed granules could sustain the maximum OLR tested. At a low OLR, these granules exhibited a loose fluffy morphology dominated by filamentous bacteria. At higher OLRs, these granules became irregularly shaped, with folds, crevices and depressions. In contrast, acetate-fed granules had a compact spherical morphology at OLRs of 6 and 9 kg COD m⁻³ d⁻¹, with better settling and strength characteristics than glucose-fed granules at similar OLRs. However, acetate-fed granules could not sustain high OLRs and disintegrated when the OLR reached 9 kg COD m⁻³ d⁻¹.
Conclusions:  The compact regular microstructure of the acetate-fed granules appeared to limit mass transfer of nutrients at an OLR of 9 kg COD m⁻³ d⁻¹. The looser filamentous microstructure of the glucose-fed granules and the subsequent irregular morphology delayed the onset of diffusion limitation and allowed significantly higher OLRs to be attained.
Significance and Impact of the Study:  High organic loading rates are possible with aerobic granules. This research would be helpful in the development of aerobic granule-based systems for high-strength wastewaters.     

Anaerobic Growth of Corynebacterium glutamicum via Mixed-Acid Fermentation

Corynebacterium glutamicum, a model organism in microbial biotechnology, is known to metabolize glucose under oxygen-deprived conditions to l-lactate, succinate, and acetate without significant growth. This property is exploited for efficient production of lactate and succinate. Our detailed analysis revealed that marginal growth takes place under anaerobic conditions with glucose, fructose, sucrose, or ribose as a carbon and energy source but not with gluconate, pyruvate, lactate, propionate, or acetate. Supplementation of glucose minimal medium with tryptone strongly enhanced growth up to a final optical density at 600 nm (OD₆₀₀) of 12, whereas tryptone alone did not allow growth. Amino acids with a high ATP demand for biosynthesis and amino acids of the glutamate family were particularly important for growth stimulation, indicating ATP limitation and a restricted carbon flux into the oxidative tricarboxylic acid cycle toward 2-oxoglutarate. Anaerobic cultivation in a bioreactor with constant nitrogen flushing disclosed that CO₂ is required to achieve maximal growth and that the pH tolerance is reduced compared to that under aerobic conditions, reflecting a decreased capability for pH homeostasis. Continued growth under anaerobic conditions indicated the absence of an oxygen-requiring reaction that is essential for biomass formation. The results provide an improved understanding of the physiology of C. glutamicum under anaerobic conditions.     

Characterization of Azo Reduction Activity in a Novel Ascomycete Yeast Strain

Several model azo dyes are reductively cleaved by growing cultures of an ascomycete yeast species, Issatchenkia occidentalis. In liquid media containing 0.2 mM dye and 2% glucose in a mineral salts base, more than 80% of the dyes are removed in 15 h, essentially under microaerophilic conditions. Under anoxic conditions, decolorization does not occur, even in the presence of pregrown cells. Kinetic assays of azo reduction activities in quasi-resting cells demonstrated the following: (i) while the optimum pH depends on dye structure, the optimum pH range was observed in the acidic range; (ii) the maximum decolorizing activity occurs in the late exponential phase; and (iii) the temperature profile approaches the typical bell-shaped curve. These results indirectly suggest the involvement of an enzyme activity in azo dye reduction. The decolorizing activity of I. occidentalis is still observed, although at a lower level, when the cells switch to aerobic respiration at the expense of ethanol after glucose exhaustion in the culture medium. Decolorization ceased when all the ethanol was consumed; this observation, along with other lines of evidence, suggests that azo dye reduction depends on cell growth. Anthraquinone-2-sulfonate, a redox mediator, enhances the reduction rates of the N,N-dimethylaniline-based dyes and reduces those of the 2-naphthol-based dyes, an effect which seems to be compatible with a thermodynamic factor. The dye reduction products were tested as carbon and nitrogen sources. 1-Amino-2-naphthol was used as a carbon and nitrogen source, and N,N-dimethyl-p-phenylenediamine was used only as a nitrogen source. Sulfanilic and metanilic acids did not support growth either as a carbon or nitrogen source.     

Effect of different carbon sources on the enhanced biological phosphorus removal in a sequencing batch reactor

The effect of the different carbon sources acetate, acetate/glucose or glucose on the enhanced biological phosphorus removal (EBPR) process was studied by experiments under alternating anaerobic–aerobic conditions in one sequencing batch reactor for each carbon source. The glucose was consumed completely within the first 30 min of the anaerobic phase whereas acetate degradation was slow and incomplete. Phosphate was released independently of the carbon source during the whole anaerobic phase. The highest phosphate release (27 mg P l⁻¹) and polyhydroxyalkanoate (PHA) storage (20 mg C g⁻¹ dry matter (DM)) during the anaerobic phase as well as the highest polyphosphate (poly-P) (8 mg P g⁻¹ DM) and glycogen storage (17 mg C g⁻¹ DM) during the aerobic phase were observed with acetate. In contrast to other investigations, glycogen storage did not increase with glucose as substrate but was significantly smaller than with acetate. The PHA composition was also influenced strongly by the carbon source. The polyhydroxyvalerate (PHV) portion of the PHA was maximal 17% for acetate and 82% for glucose. Due to the strong influence of the carbon source on the PHA concentration and composition, PHA storage seems to regulate mainly the phosphate release and uptake. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of ethanol by filamentous and yeast-like forms of <Emphasis Type="Italic">Mucor indicus</Emphasis> from fructose,glucose, sucrose,and molasses

The fungus Mucor indicus is found in this study able to consume glucose and fructose, but not sucrose in fermentation of sugarcane and sugar beet molasses. This might be an advantage in industries which want to selectively remove glucose and fructose for crystallisation of sucrose present in the molasses. On the other hand, the fungus assimilated sucrose after hydrolysis by the enzyme invertase. The fungus efficiently grew on glucose and fructose and produced ethanol in synthetic media or from molasses. The cultivations were carried out aerobically and anaerobically, and manipulated toward filamentous or yeast-like morphology. Ethanol was the major metabolite in all the experiments. The ethanol yield in anaerobic cultivations was between 0.35 and 0.48 g/g sugars consumed, depending on the carbon source and the growth morphology, while a yield of as low as 0.16 g/g was obtained during aerobic cultivation. The yeast-like form of the fungus showed faster ethanol production with an average productivity of 0.90 g/l h from glucose, fructose and inverted sucrose, than the filamentous form with an average productivity of 0.33 g/l h. The biomass of the fungus was also analyzed with respect to alkali-insoluble material (AIM), chitin, and chitosan. The biomass of the fungus contained per g maximum 0.217 g AIM and 0.042 g chitosan in yeast-like cultivation under aerobic conditions.     

Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production.     

Pyruvate production by Enterococcus casseliflavus A-12 from gluconate in an alkaline medium

A newly isolated lactic acid bacterium, Enterococcus casseliflavus A-12, produced pyruvic acid (16 g/l) during aerobic culture in an alkaline medium containing sodium gluconate (50 g/l) as the carbon source. The production was dependent on the pH of the culture, the optimum initial pH being 10.0. With static culture, the organism produced lactic acid (2.7 g/l) from both gluconate and glucose. Pyruvate did not accumulate in growing cultures on glucose, but resting cells obtained from a culture on gluconate produced pyruvate from glucose as well as gluconate. The enzyme profiles of the organism, which grew on gluconate and glucose, suggested that gluconate was metabolized via the Entner-Doudoroff and Embdem-Meyerhof-Parnas pathways in aerobic culture, and that glucose was oxidized mainly via the latter pathway under both aerobic and anaerobic conditions. Gluconokinase, a key enzyme in the aerobic metabolism of gluconate, was partially purified from this strain and characterized.     

Isolation and characterization of a novel facultatively alkaliphilic bacterium,Corynebacterium sp., grown on n-alkanes

A novel facultatively alkaliphilic bacterium that grows on a chemically defined medium containing n-alkanes as the sole carbon source was isolated from soil. The isolate was obligately aerobic, non-motile, gram-positive, and formed metachromatic granules. It was not acidfast and did not form endospores. The cell wall contained meso-diaminopimelic acid, arabinose, and galactose; the glycan moiety of the cell wall contained acetyl residues. The bacterium was catalase-positive, oxidasenegative, and the G+C content of DNA was 70.8 mol%. According to these tests, the isolate was assigned to the genus Corynebacterium. The bacterium grew well between pH 6.2 to 10.2 and the doubling time in this pH range was 4–6 h. For the growth of the isolate, added Na⁺ in the culture medium stimulated growth, but was not indispensable at both pH 7.2 and pH 10.2. In addition to hydrocarbons, the isolate was able to grow on a chemically defined medium containing acetate, glucose, or fructose as the sole carbon source. Analysis of reduced minus oxidized difference spectra of whole cells showed that the bacterium only possessed less than one tenth the amount of total cytochromes as compared with Bacillus alcalophilus. The above results sugest that the bacterium has characteristics different than those of the alkaliphilic Bacillus previously described.     

Oxygen- and Glucose-Dependent Regulation of Central Carbon Metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose⁻¹) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.     

Production of biomass and filamentous hemagglutinin by Bordetella bronchiseptica

The mammalian pathogen Bordetella bronchiseptica was grown under controlled batch conditions with glutamate as the primary carbon and nitrogen source. First, a Box-Behnken statistical design quantified the effect of Mg, sulfate, and nicotinate on the antigen filamentous hemagglutinin (FHA) formation. Using lactic acid as a secondary carbon source for pH control, Mg, and SO₄ each negatively affected antigen expression, while nicotinate positively affected antigen expression. Sulfate had a stronger negative effect than Mg with 10 mM eliminating FHA altogether; the highest FHA expression (about 1,000 ng/mL) occurred when either Mg concentration or SO₄ concentration, but not both, was about 0.1 mM. Using two Mg and SO₄ compositions modeled to yield the greatest antigen expression, three other organic acids were compared as the secondary carbon source: acetate, citrate, and succinate. Mixtures of acetate and glutamate resulted in the greatest organic acid consumption, OD, and FHA concentration (about 1,500 ng/mL), although significant acetate accumulated during these batch processes. The mechanism leading to elevated FHA expression when acetate is the secondary carbon source is unknown, particularly since these cultures were most prone to phase shift to Bvg^? cultures.     

Changes in the microbial community structure of filaments and floc formers in response to various carbon sources and feeding patterns

Filamentous bulking is a complicated problem in wastewater treatment plants treating various wastewaters, leading to the deterioration of the settling properties and the effluent quality. This study systematically investigated long-term effects of various carbon sources and feeding patterns on the growth of filamentous bacteria, in order to reveal the mechanism of filamentous bulking. Sludge volume index (SVI), microscopic observations, staining (Gram and Neisser staining), scan electron microscopic, and fluorescent in situ hybridization (FISH) were used to monitor the bulking and track the changes of microbial morphology and community structure of activated sludge in six lab-scale sequencing batch reactors (SBRs) fed with different carbon sources. Filamentous bulking was not observed in all SBRs under anoxic feeding pattern with a short fill time, in which SVI remained below 150 mL/g. In contrast, serious bulking (SVI?>?500 mL/g) occurred under aerobic feeding pattern when fed with ethanol, propionate, acetate, and glucose, in which Thiothrix and Sphaerotilus natans proliferated as dominant filaments. Compared to glucose-fed reactor, relatively light bulking was caused in starch-fed reactor with the growth of Nostocoida limicola II. In addition, flocs in starch-fed reactor were more open and fluffy than flocs formed on readily biodegradable substrates. Finally, a framework integrating kinetic selection, diffusion selection, storage selection, and protozoa capture mechanism was proposed to explain filamentous bulking.     

Inoculum Size Effect in Dimorphic Fungi: Extracellular Control of Yeast-Mycelium Dimorphism in Ceratocystis ulmi

We studied the inoculum size effect in Ceratocystis ulmi, the dimorphic fungus that causes Dutch elm disease. In a defined glucose-proline-salts medium, cells develop as budding yeasts when inoculated at ≥10⁶ spores per ml and as mycelia when inoculated at <10⁶ spores per ml. The inoculum size effect was not influenced by inoculum spore type, age of the spores, temperature, pH, oxygen availability, trace metals, sulfur source, phosphorous source, or the concentration of glucose or proline. Similarly, it was not influenced by added adenosine, reducing agents, methyl donors, amino sugars, fatty acids, or carbon dioxide. Instead, growing cells excreted an unknown quorum-sensing factor that caused a morphological shift from mycelia to budding yeasts. This yeast-promoting effect is abolished if it is extracted with an organic solvent such as ethyl acetate. The quorum-sensing activity acquired by the organic solvent could be added back to fresh medium in a dose-dependent fashion. The quorum-sensing activity in C. ulmi spent medium was specific for C. ulmi and had no effect on the dimorphic fungus Candida albicans or the photomorphogenic fungus Penicillium isariaeforme. In addition, farnesol, the quorum-sensing molecule produced by C. albicans, did not inhibit mycelial development of C. ulmi when present at concentrations of up to 100 μM. We conclude that the inoculum size effect is a manifestation of a quorum-sensing system that is mediated by an excreted extracellular molecule, and we suggest that quorum sensing is a general phenomenon in dimorphic fungi.     

Influence of Acidic pH on Hydrogen and Acetate Production by an Electrosynthetic Microbiome

Production of hydrogen and organic compounds by an electrosynthetic microbiome using electrodes and carbon dioxide as sole electron donor and carbon source, respectively, was examined after exposure to acidic pH (∼5). Hydrogen production by biocathodes poised at −600 mV vs. SHE increased>100-fold and acetate production ceased at acidic pH, but ∼5–15 mM (catholyte volume)/day acetate and>1,000 mM/day hydrogen were attained at pH ∼6.5 following repeated exposure to acidic pH. Cyclic voltammetry revealed a 250 mV decrease in hydrogen overpotential and a maximum current density of 12.2 mA/cm² at −765 mV (0.065 mA/cm² sterile control at −800 mV) by the Acetobacterium-dominated community. Supplying −800 mV to the microbiome after repeated exposure to acidic pH resulted in up to 2.6 kg/m³/day hydrogen (≈2.6 gallons gasoline equivalent), 0.7 kg/m³/day formate, and 3.1 kg/m³/day acetate ( = 4.7 kg CO₂ captured).     

Effect of growth conditions on the molecular weight of poly-3-hydroxybutyrate produced by <Emphasis Type="Italic">Azotobacter chroococcum</Emphasis> 7B

It has been shown that poly-3-hydroxybutyrate (PHB) of predetermined molecular weight can be obtained by varying the growth conditions of the producer strain, Azotobacter chroococcum 7B: pH, temperature, aeration, presence of sodium acetate as an additional carbon source, or growth on crude complex carbon sources (molasses, vinasse, or starch). High-molecular-weight polymer can be obtained at pH 7.0, optimal for the culture (1485 kDa), temperature 30–37°C (1600–1450 kDa, respectively), and low aeration (2215 kDa). The following factors decrease PHB MW: pH deviation to the acidic (pH 6.0, 476 kDa) or alkaline (pH 8.0, 354 kDa) range or lower temperature (20°C, 897 kDa). Introduction of additional carbon source (sodium acetate) at concentrations in the medium varying from 0 to 5 g/l provides an original method of production of PHB with predetermined MW in a wide range, from 270 to 1515 kDa, with high PHB content in the cell.     

Degradation pathway, toxicity and kinetics of 2,4,6-trichlorophenol with different co-substrate by aerobic granules in SBR

The present study deals with cultivation of 2,4,6-trichlorophenol (TCP) degrading aerobic granules in two SBR systems based on glucose and acetate as co-substrate. Biodegradation of TCP containing wastewater starting from 10 to 360 mg L⁻¹ with more than 90% efficiency was achieved. Sludge volume index decreases as the operation proceeds to stabilize at 35 and 30 mL g⁻¹ while MLVSS increases from 4 to 6.5 and 6.2 g L⁻¹ for R1 (with glucose as co-substrate) and R2 (with sodium acetate as co-substrate), respectively. FTIR, GC and GC/MS spectral studies shows that the biodegradation occurred via chlorocatechol pathway and the cleavage may be at ortho-position. Haldane model for inhibitory substrate was applied to the system and it was observed that glucose fed granules have a high specific degradation rate and efficiency than acetate fed granules. Genotoxicity studies shows that effluent coming from SBRs was non-toxic.     

Characterization of Exopolysaccharides Produced by Plant-Associated Fluorescent Pseudomonads

A total of 214 strains of plant-associated fluorescent pseudomonads were screened for the ability to produce the acidic exopolysaccharide (EPS) alginate on various solid media. The fluorescent pseudomonads studied were saprophytic, saprophytic with known biocontrol potential, or plant pathogenic. Approximately 10% of these strains exhibited mucoid growth under the conditions used. The EPSs produced by 20 strains were isolated, purified, and characterized. Of the 20 strains examined, 6 produced acetylated alginate as an acidic EPS. These strains included a Pseudomonas aeruginosa strain reported to cause a dry rot of onion, a strain of P. viridiflava with soft-rotting ability, and four strains of P. fluorescens. However, 12 strains of P. fluorescens produced a novel acidic EPS (marginalan) composed of glucose and galactose (1:1 molar ratio) substituted with pyruvate and succinate. Three of these strains were soft-rotting agents. Two additional soft-rotting strains of P. fluorescens produced a third acidic novel EPS composed of rhamnose, mannose, and glucose (1:1:1 molar ratio) substituted with pyruvate and acetate. When sucrose was present as the primary carbon source, certain strains produced the neutral polymer levan (a fructan) rather than an acidic EPS. Levan was produced by most strains capable of synthesizing alginate or the novel acidic EPS containing rhamnose, mannose, and glucose but not by strains capable of marginalan production. It is now evident that the group of bacteria belonging to the fluorescent pseudomonads is capable of elaborating a diverse array of acidic EPSs rather than solely alginate.     

Influence of cultivation conditions on production of lignocellulolytic enzymes by Ceriporiopsis subvermispora

The aim of this work was to make a survey describing factors that influence the production of extracellular enzymes by white-rot fungus Ceriporiopsis subvermispora responsible for the degradation of lignocellulolytic materials. These factors were: carbon sources (glucose, cellulose, hemicellulose, lignin, maltose and starch), nitrogen sources (ammonium sulphate, potassium nitrate, urea, albumin and peptone), pH, temperature and addition of three different concentrations of Cu²⁺ and Mn²⁺. The cellulase and xylanase activities were similar in medium with different carbon sources and the highest cellulase and xylanase activities were measured in medium with urea and potassium nitrate as nitrogen sources, respectively. The highest laccase activity was observed in medium with lignin and peptone as carbon and nitrogen sources. In other experiments, time course of production of lignocellulolytic enzymes by white-rot fungus C. subvermispora in medium with lignin or glucose as carbon sources was observed.     

Oxygen diffusion and consumption in active aerobic granules of heterogeneous structure

The interior structure of aerobic granules is highly heterogeneous, hence, affecting the transport and reaction processes in the granules. The granule structure and the dissolved oxygen profiles were probed at the same granule in the current work for possible estimation of transport and kinetic parameters in the granule. With the tested granules fed by phenol or acetate as carbon source, most inflow oxygen was consumed by an active layer thickness of less than 125 μm on the granule surface. The confocal laser scanning microscopy scans also revealed a surface layer thickness of approximately 100 μm consisting of cells. The diffusivities of oxygen transport and the kinetic constant of oxygen consumption in the active layers only were evaluated. The theoretical models adopted in literature that ignored the contributions of the layered structure of aerobic granule could have overlooked the possible limitations on oxygen transport.