Growth kinetics of mixed culture in salmonella enrichment media

A technique for investigating the kinetics of salmonella enrichment is reported. Its use with four enrichment media (Rappaport's medium, Muller-Kauffmann tetrathionate broth (MKT) tetrathionate broth and selenite F) is described and the effect of elevated temperature on the growth kinetics shown. Rappaport's medium at 37 degrees C and MKT at either 37 degrees C or 42 degrees C were far superior to selenite F and tetrathionate broth in their selective properties and, with the exception of Rappaport's medium, the use of elevated temperature increased the selectivity of the media.     

No evidence for the biodegradation of benzotriazole by elective culture or continuous enrichment

Summary Elective cultures and continuous enrichment failed to demonstrate biodegradation of benzotriazole. It is considered that a resistance to degradation in the environment could be expected.     

Reduction of perchlorate by an anaerobic enrichment culture

Summary A mixed bacterial culture capable of reducing perchlorate stoichiometrically to chloride under naerobic conditions was enriched from municipal digester sludge. The reduction of 10 mM perchlorate resulted in oxidation of the medium and cessation of perchlorate reduction. The activity was recovered on addition of a reducing agent. Addition of air to the culture during perchlorate reduction immediately terminated the process and aeration for 12 h permanently destroyed the ability of the culture to reduce perchlorate. The culture also reduced nitrite, nitrate, chlorite, chlorate and sulfate. The presence of 10 mM nitrite or chlorite completely inhibited perchlorate reduction, whereas the same concentration of chlorate decreased the reduction rate. Nitrate or sulfate did not affect perchlorate reduction. Chlorate and chlorite, suspected intermediates in the reduction of perchlorate to chloride, were not detected in any cultures during reduction of perchlorate.     

Anaerobic degradation of chlorophenol by an enrichment culture

Summary An anaerobic mixed culture from sewage sludge was enriched in a yeast extract and peptone-containing medium; it was able to degrade 2-cholorophenol completely to methane and CO₂. Degradation rates of 2-chlorophenol of up to 0.18 g/l per day were observed in suspended cultures without biomass retention and of 0.375 g/l per day in cultures immobilized on Liapor clay beads. Attempts to isolate the dechlorinating organism failed. The mixed culture was reduced to three morphologically distinctive microorganisms using a medium with limited amounts of yeast extract and peptone and n-butyrate as a co-substrate. Under these conditions the phenol-degrading bacterium was lost and phenol accumulated in the medium. No growth and no dehalogenation of 2-chlorophenol was obtained when yeast extract and peptone were omitted completely. Besides serving as a source of supplementary components, yeast extract and peptone were apparently required as the main source of carbon, wereas reducing equivalents for reductive dehalogenation were obtained by oxidation of n-butyrate. A spirochaete-like organism was presumably the dechlorinating bacterium. The mixed culture lost its dehalogenation capability if this organism was lost. n-Butyrate could be replaced by n-valerate, hexanoate, heptanoate, octanoate, pelargonic acid, n-decanoic acid or palmitate as co-substrates for dehalogenation of either 2-chlorophenol, 2-bromophenol or complete dechlorination of 2,6-dichlorophenol, whereas from 2,4-dichlorophenol only the substituent in the ortho-position could be eliminated.Dedicated to Professor O. Kandler on the occassion of his 70th birthdayOffprint requests to: J. Winter     

Methanogenic degradation of acetone by an enrichment culture

An anaerobic enrichment culture degraded 1 mol of acetone to 2 mol of methane and 1 mol of carbon dioxide. Two microorganisms were involved in this process, a filament-forming rod similar to Methanotrix sp. and an unknown rod with round to slightly pointed ends. Both organisms formed aggregates up to 300 m in diameter. No fluorescing bacteria were observed indicating that hydrogen or formate-utilizing methanogens are not involved in this process. Acetate was utilized in this culture by the Methanothrix sp. Inhibition of methanogenesis by bromoethanesulfonic acid or acetylene decreased the acetone degradation rate drastically and led to the formation of 2 mol acetate per mol of acetone. Streptomycin completely inhibited acetone degradation, and neither acetate nor methane was formed. ¹⁴CO₂ was incorporated exclusively into the C-1 atom of acetate indicating that acetone is degraded via carboxylation to an acetoacetate residue. It is concluded that acetone is degraded by a coculture of an eubacterium and an acetate-utilizing methanogen and that acetate is the only intermediate transferred between both. The energetical problems of the eubacterium converting acetone to acetate are discussed.     

Methanogenic fermentation of benzoate in an enrichment culture

Enrichment cultures inoculated with black mud fermented benzoate according to the stoichiometric equation: 4 C₆H₅CO₂H+18 H₂O 15 CH₄+13 CO₂.Trans-2-hydroxycyclohexanecarboxylate, 2-oxo-cyclohexanecarboxylate, pimelate, caproate, butyrate, acetate, and molecular hydrogen were shown to be regular components of the culture fluid occurring in low concentrations. Inhibition of methanogenesis by chloroform, 4-chlorobutyrate, or 2-bromooctanoate resulted in a cessation of the benzoate breakdown after all intermediates had accumulated. It is proposed that benzoate is fermented via a direct reductive pathway to butyrate, acetate, H₂, and CO₂, whereafter butyrate is converted to acetate and H₂, and the latter substrates are fermented to CH₄ and CO₂ by methane producers.     

Pentachlorophenol biodegradation by Pseudomonas spp. UG25 and UG30

Eighty-nine bacterial isolates obtained by enrichment from pentachlorophenol (PCP)-contaminated soil samples were tested for PCP dechlorination activity and hybridization to pcpB (encoding PCP-4-monooxygenase) and pcpC (encoding tetrachlorohydroquinone reductive dehalogenase) gene probes synthesized by polymerase chain reaction from Flavobacterium sp. ATCC 39723 genomic DNA. Seven isolates were able to dechlorinate PCP, hybridize to both pcpB and pcpC DNA probes, and mineralize sodium pentachlorophenate (NaPCP) at an initial concentration of 100g/ml. Although the seven PCP-mineralizing isolates possessed DNA sequences homologous to the Flavobacterium pcpB and pcpC genes, restriction analysis revealed sequence differences between the isolates and the Flavobacterium PCP dechlorination genes. Two isolates, designated UG25 and UG30, with the fastest onset and highest extent of PCP mineralization were selected for further study. Both isolates were tentatively identified as Pseudomonas spp. and exhibited stoichiometric release of Cl– ions as PCP was degraded. The release of Cl– began concomitantly with PCP disappearance from the medium. Both UG25 and UG30 degraded NaPCP at a concentration of 250 g/ml in a minimal salt medium. Supplementation of the medium with glutamate increased the NaPCP degradation threshold of UG25 to a concentration of 300 g/ml but did not affect that of UG30. 31P-NMR spectra of UG25 and UG30 cell suspensions exposed to PCP showed lower intracellular ATP levels and a more acidic cytoplasmic pH relative to untreated cells. This de-energization may explain the lack of cell growth in the presence of high PCP concentrations.     

Pentachlorophenol degradation: a pure bacterial culture and an epilithic microbial consortium

The steady-state growth of a Flavobacterium strain known to utilize pentachlorophenol (PCP) was examined when cellobiose and PCP simultaneously limited its growth rate in continuous culture. A concentration of 600 mg of PCP per liter in influent medium could be continuously degraded without affecting steady-state growth. We measured specific rates of PCP carbon degradation as high as 0.15 +/- 0.01 g (dry weight) of C per h at a growth rate of 0.045 h-1. Comparable specific rates of PCP degradation were obtained and maintained by PCP-adapted, natural consortia of epilithic microorganisms. The consortium results suggest that a fixed-film bioreactor containing a PCP-adapted natural microbial population could be used to treat PCP-contaminated water.     

Short-term changes in phosphorus storage in an oligotrophic Everglades wetland ecosystem receiving experimental nutrient enrichment

Natural, unenriched Evergladeswetlands are known to be limited by phosphorus(P) and responsive to P enrichment. However,whole-ecosystem evaluations of experimental Padditions are rare in Everglades or otherwetlands. We tested the response of theEverglades wetland ecosystem to continuous,low-level additions of P (0, 5, 15, and30 g L^–1 above ambient) in replicate,100 m flow-through flumes located in unenrichedEverglades National Park. After the first sixmonths of dosing, the concentration andstanding stock of phosphorus increased in thesurface water, periphyton, and flocculentdetrital layer, but not in the soil or macrophytes. Of the ecosystem components measured, total P concentration increased the most in the floating periphyton mat (30 g L^–1: mean = 1916 g P g^–1, control: mean =149 g P g^–1), while the flocculentdetrital layer stored most of the accumulated P(30 g L^–1: mean = 1.732 g P m^–2,control: mean = 0.769 g P m^–2). Significant short-term responsesof P concentration and standing stock wereobserved primarily in the high dose (30 gL^–1 above ambient) treatment. Inaddition, the biomass and estimated P standingstock of aquatic consumers increased in the 30and 5 g L^–1 treatments. Alterationsin P concentration and standing stock occurredonly at the upstream ends of the flumes nearestto the point source of added nutrient. Thetotal amount of P stored by the ecosystemwithin the flume increased with P dosing,although the ecosystem in the flumes retainedonly a small proportion of the P added over thefirst six months. These results indicate thatoligotrophic Everglades wetlands respondrapidly to short-term, low-level P enrichment,and the initial response is most noticeable inthe periphyton and flocculent detrital layer.     

Pentachlorophenol degradation: a pure bacterial culture and an epilithic microbial consortium.

The steady-state growth of a Flavobacterium strain known to utilize pentachlorophenol (PCP) was examined when cellobiose and PCP simultaneously limited its growth rate in continuous culture. A concentration of 600 mg of PCP per liter in influent medium could be continuously degraded without affecting steady-state growth. We measured specific rates of PCP carbon degradation as high as 0.15 +/- 0.01 g (dry weight) of C per h at a growth rate of 0.045 h-1. Comparable specific rates of PCP degradation were obtained and maintained by PCP-adapted, natural consortia of epilithic microorganisms. The consortium results suggest that a fixed-film bioreactor containing a PCP-adapted natural microbial population could be used to treat PCP-contaminated water.     

Mesocosm studies on the influence of phosphate enrichment on ammonium and nitrate flux in an oligotrophic lake

The fate of ¹⁵NH _inf4 ^sup+ and ¹⁵NOinf3⁻ was followed in control and PO _inf4 ^sup3− enriched 1570 l mesocosms filled with epilimnetic water from an oligotrophic Rocky Mountain lake. Volumetric incorporation of ¹⁵NH _inf4 ^sup+ and ¹⁵NO _inf3 ^sup− into phytoplankton and bacterioplankton (particulates between 280 and 0.7 μm), and crustacean zooplankton > 80 μm was enhanced by PO _inf4 ^sup3− , but no increase in biomass specific rates of uptake by phytoplankton and bacteria occurred for either form of ¹⁵N. Dilution of both ¹⁵NH _inf4 ^sup+ and ¹⁵NO _inf3 ^sup− by ¹⁴NH _inf4 ^sup+ and ¹⁴NO _inf3 ^sup− , respectively, was evident indicating regeneration of these nutrients, but regeneration rates were not effected by PO _inf4 ^sup3− enrichment. The results illustrate the strong trophic coupling between N dynamics and PO _inf4 ^sup3− enrichment in this system.     

Organic enrichment of sediments reduces arbuscular mycorrhizal fungi in oligotrophic lake plants

1. Arbuscular mycorrhizal fungi (AMF) commonly colonise isoetid species inhabiting oxygenated sediments in oligotrophic lakes but are usually absent in other submerged plants. We hypothesised that organic enrichment of oligotrophic lake sediments reduces AMF colonisation and hyphal growth because of sediment O₂ depletion and low carbon supply from stressed host plants. 2. We added organic matter to sediments inhabited by isoetids and measured pore‐water chemistry (dissolved O₂, inorganic carbon, Fe²⁺ and ), colonisation intensity of roots and hyphal density after 135 days of exposure. 3. Addition of organic matter reduced AMF colonisation of roots of both Lobelia dortmanna and Littorella uniflora, and high additions stressed the plants. Even small additions of organic matter almost stopped AMF colonisation of initially un‐colonised L. uniflora, though without reducing plant growth. Mean hyphal density in sediments was high (6 and 15 m cm^?3) and comparable with that in terrestrial soils (2–40 m cm^?3). Hyphal density was low in the upper 1 cm of isoetid sediments, high in the main root zone between 1 and 8 cm and positively related to root density. Hyphal surface area exceeded root surface area by 1.7–3.2 times. 4. We conclude that AMF efficiently colonise isoetids in oligotrophic sediments and form extensive hyphal networks. Small additions of organic matter to sediments induce sediment anoxia and reduce AMF colonisation of roots but cause no apparent plant stress. High organic addition induces night‐time anoxia in both the sediment and the plant tissue. Tissue anoxia reduces root growth and AMF colonisation, probably because of restricted translocation of nutrient ions and organic solutes between roots and leaves. Isoetids should rely on AMF for P uptake on nutrient‐poor mineral sediments but are capable of growing without AMF on organic sediments.     

Effects of oxygen on aerobic solid-state biodegradation kinetics

Oxygen is a critical control variable for composting and other solid-state biodegradation processes. In this study we examined the effect of varying oxygen concentrations (1%, 4%, and 21% O2 (v/v)) on biodegradation kinetics under different substrate (sewage sludge and synthetic food waste), temperature (35, 45, 55, and 65 degrees C), and moisture (36-60% H2O) conditions. Three forms of a saturation or Monod-type model and one form of an exponential model were evaluated against data from extensive experiments under carefully controlled environmental conditions. The exponential model performed well at temperatures from 35 to 55 degrees C but had problems at higher temperatures. The Monod-type models yielded the best fit based on R2 values. Multiple linear regression was used to express the oxygen half-saturation coefficient as a function of temperature and moisture. For a modified one-parameter saturation model the half-saturation coefficient varied from -0.67% to 1.74% v/v O2 under the range of conditions typical of composting systems. While the positive correlation of biodegradation rate with oxygen concentration reported by previous researchers held true for temperatures below 55 degrees C, an inverse relationship was found at 65 degrees C. Although this study did not directly examine anaerobic conditions, the results under microaerophilic conditions suggest oxygen may not offer kinetic advantages for extreme thermophilic biodegradation processes.     

Consecutive reaction kinetics involving distributed fraction of methanogens in fluidized-bed bioreactors

A kinetic model involving the distributed fractions of acidogens and methanogens is proposed. To determine the fluxes and biochemical reaction rates of the substrate sucrose and its intermediates, volatile fatty acids (VFAs) in bulk liquid and within the biofilm, a kinetic model was developed by combining the solid-phase model with the liquid-phase model. The predicted substrate removal efficiencies of the conventional and tapered fluidized-bed bioreactors (CFB, TFBs) are in good agreement with the experimental results. The biofilm thickness in TFBs are thicker than that in CFB, resulting in performance enhancement with TFBs. The simulated results obtained from the kinetic model show that methanogenesis is the rate-limiting step of degradation of the simple organic compound (sucrose), and the chemical oxygen demand (COD) concentration in the effluent is mainly contributed by the intermediates VFAs. The distributed fractions of acidogens and methanogens determined experimentally are 0.4 and 0.6, respectively.     

Cooperative catabolic pathways within an atrazine-degrading enrichment culture isolated from soil

Atrazine degradation previously has been shown to be carried out by individual bacterial species or by relatively simple consortia that have been isolated using enrichment cultures. Here, the degradative pathway for atrazine was examined for a complex 8-membered enrichment culture. The species composition of the culture was determined by PCR-DGGE. The bacterial species included Agrobacterium tumefaciens, Caulobacter crescentus, Pseudomonas putida, Sphingomonas yaniokuyae, Nocardia sp., Rhizobium sp., Flavobacterium oryzihabitans, and Variovorax paradoxus. All of the isolates were screened for the presence of known genes that function for atrazine degradation including atzA,-B,-C,-D,-E,-F and trzD,-N. Dechlorination of atrazine, which was obligatory for complete mineralization, was carried out exclusively by Nocardia sp., which contained the trzN gene. Following dechlorination, the resulting product, hydroxyatrazine was further degraded via two separate pathways. In one pathway Nocardia converted hydroxyatrazine to N-ethylammelide via an unidentified gene product. In the second pathway, hydroxyatrazine generated by Nocardia sp. was hydrolyzed to N-isopropylammelide by Rhizobium sp., which contained the atzB gene. Each member of the enrichment culture contained atzC, which is responsible for ring cleavage, but none of the isolates carried the atzD,-E, or -F genes. Each member further contained either trzD or exhibited urease activity. The enrichment culture was destabilized by loss of Nocardia sp. when grown on ethylamine, ethylammelide, and cyanuric acid, after which the consortium was no longer able to degrade atrazine. The analysis of this enrichment culture highlights the broad level bacterial community interactions that may be involved in atrazine degradation in nature.     

Evaporation kinetics of polychorinated biphenyls during biodegradation experiments

Summary During microbial degradation of polychlorinated biphenyls (PCB) in liquid media two processes take place simultaneously: elimination of PCB and evaporation of PCB. The physical loss of PCB due to evaporation causes frequently false positive results in long-term biodegradation experiments. Therefore, if only the PCB concentration is to be measured, its determination in both liquid and gaseous phase is essential for a correct appraisal of biodegradation. The kinetics of PCB evaporation have been monitored and the evaporation rate constants for individual PCB congeners have been determined. The values of evaporation rate constants show a good corelation with the values of the 1-octanol/water partition coefficient.     

Growth kinetics of EDTA biodegradation by Burkholderia cepacia

A pure culture of an EDTA-degrading strain was isolated from the Taiwan environment. It was identified as Burkholderia cepacia, an aerobic bacterium, elliptically shaped with a length of 5–15 m. The degradation assay showed that the degradation efficiency of Fe-EDTA by B. cepacia was approximately 91%. Evaluation of kinetic parameters showed that Fe-EDTA degradation followed substrate inhibition kinetics. This is evident from the decrease in specific growth rate with an increase in the initial substrate concentration greater than 500 mg/l. To estimate the kinetic parameters – _max, K_S and K_I, five substrate–inhibition models were used. From the results of non-linear regression, the value of _max ranged from 0.150 to 0.206 d^–1, K_S from 74 to 87 mg/l, and K_I from 890 to 2289 mg/l. The five models were found to underestimate the maximum specific growth rate by 1.5–3.7. Therefore, predictions based on these models would result in lower predicted value than those from the experimental kinetic data.     

Modeling the temperature kinetics of aerobic solid-state biodegradation

This study evaluated three models of microbial temperature kinetics using CO2 respiration data from aerobic solid-state biodegradation experiments. The models included those of Andrews and Kambhu/Haug, Ratkowsky et al., and the Cardinal Temperature Model with Inflection (CTMI) of Rosso et al. A parameter estimation routine implemented the Complex-Box search method for each model on 48 data sets collected during the composting of synthetic food waste or sewage-sludge (biosolids) mixed with maple wood chips at different oxygen concentrations and extents of decomposition. Each of the three nonlinear temperature kinetic functions proved capable of modeling a wide range of experimental data sets. However, the models differed widely in the consistency of their parameters. Parameters in the CTMI model were more stable over the course of the degradation process, and that variability which did arise was directly related to changes in the microbial process. Additional benefits of the CTMI model include the ease of parameter determinations, which can be approximated directly from laboratory experiments or full-scale system analysis, and the direct value of its parameters in engineering design and process control under a wide range of biodegradation conditions.     

Fermentative degradation of acetone by an enrichment culture in membrane-separated culture devices and in cell suspensions

Abstract We have recently demonstrated that cultured human intestinal HT-29 and Caco-2 cell lines express receptors for the F1845 fimbrial adhesin harbored by the diarrheagenic C1845 Escherichia coli (Kernéis et al., Infect. Immun. 59 (1991) 4013–4018). This adhesinn belongs to a family of adhesins including the Dr hemagglutinin and the afimbrial adhesin AFA-1 harbored by uropathogenic E. coli . Here we investigated the cell association of laboratory E. coli strains expressing the Dr hemagglutinin and the afimbrial adhesin AFA-I with human cultured enterocyte-like or mucosecreting cells. We observed that the E. coli strains bearing these adhesins adhere both to human intestinal undifferentiated and differentiated fluid-transporting cells, and to mucus-secreting cells. This result strongly suggests a high capacity of intestinal colonization for the uropathogenic E. coli harboring adhesive factors belonging to the Dr adhesin family. These results further corroborate the intestinal colonization by uropathogenic E. coli of the Dr family related to the fecal-perineal-urethral hypothesis of urinary tract infection pathogenesis.