Quantitative selection of denitrifying bacteria in continuous cultures and requirement for organic carbon. I. Starch

A mixed population of bacteria from bottom sludge of nitrogen wastewater reservoir was incubated in continuous culture in medium containing 1000 mg nitrate nitrogen/l and starch. Maximal efficiency of denitrification was 5 mg N/l/h. Marked changes in participation of denitrifying bacteria (16-76%) among total number of bacteria was observed, this being dependent on the ratio of starch concentration (CS) to nitrogen concentration (CN) in the medium. The optimal CS/CN ratio ensuring highest participation of denitrifying bacteria was 3.2. The amount of starch required for the denitrification of a defined quantity of nitrogen is negatively correlated (r = -0.98) with the frequency of the occurrence of denitrifying bacteria (XD) and is: CS = (5.53-0.028XD) CN. The denitrifying bacteria in continuous culture were dominated, depending on CS/CN ratio and flow rate of medium, by Alcaligenes faecalis, Paracoccus denitrificans or Pseudomonas mendocina, that is species unable to hydrolyse starch.     

Effect of various sources of organic carbon and high nitrite and nitrate concentrations on the selection of denitrifying bacteria. II. Continuous cultures in packed bed reactors

The effect of different organic compounds, nitrites and nitrates at the concentration of 1,000 mg N/l on the quantitative and strain-specific selection of denitrifying bacteria was determined in anaerobic packed bed reactors. Both the source of carbon and nitrogen form influenced strain specificity and the frequency of occurrence of denitrifying bacteria. The frequency of denitrifying bacteria within packed bed reactor ranged in different media from 11% (glucose and nitrates) to 100% (methanol and ethanol with nitrates). A single species selection was observed in the presence of nitrites within packed bed reactor: Pseudomonas aeruginosa in medium with acetate. Pseudomonas stutzeri in medium with ethanol, Pseudomonas mendocina in medium with methanol and Pseudomonas fluorescens in medium with glucose. When nitrates were present in packed bed reactor, the dominating bacteria were: P. stutzeri in medium with acetate, P. fluorescens in medium with ethanol, Paracoccus denitrificans in medium with methanol and Alcaligenes faecalis in medium with glucose.     

Biotransformation of phosphogypsum in media containing different forms of nitrogen

Studies on the biotransformation of phosphogypsum (a waste product formed in the course of the production of phosphorous fertilizers) with the use of sulfate reducing bacteria (SRB) demonstrated that it is a good source of sulfates and biogenic elements for these bacteria, though the addition of organic carbon and nitrogen is necessary. The aim of this study was to investigate the form of nitrogen and C:N ratio in the medium on the growth of SRB community in cultures containing phosphogypsum. Batch community cultures of sulfate reducing bacteria were maintained in medium with phosphogypsum (5.0 g/l), different concentrations of sodium lactate (1.6 - 9.4 g/l) and different forms (NH4CI, CO(NH2)2, KNO3) and concentrations (0 - 250 mg/l) of nitrogen. The growth of SRB was studied in the C:N ratio of from 2:1 to 300:1. It was found that: 1 - the best source of nitrogen for SRB is urea, followed by ammonium, the worst were nitrates; 2 - the bacteria were also able to grow in medium without nitrogen but their activity was then by approximately 15% lower than in optimal growth conditions; 3 - in medium with KNO3 inhibition of sulfate reduction by approx. 50% was observed; 4 - the highest reduction of nitrates (removal of nitrate) in media with phosphogypsum and nitrates was at limiting concentrations of sodium lactate. This is probably caused by the selection under these conditions (low concentration of hydrogen sulfide) of denitrifying bacteria or sulfate reducing bacteria capable of using nitrates as an electron acceptor.     

Development of an improved chemically defined minimal medium for Listeria monocytogenes.

A chemically defined minimal medium for Listeria monocytogenes has been developed by modification of Welshimer's medium. The growth factors required by L. monocytogenes Scott A are leucine, isoleucine, arginine, methionine, valine, cysteine (each at 100 mg/liter), riboflavin and biotin (each at 0.5 micrograms/ml), thiamine (1.0 micrograms/ml), and thioctic acid (0.005 micrograms/ml). Growth was stimulated by 20 micrograms of Fe3+ per ml as ferric citrate. Glucose (1%) and glutamine (600 mg/liter) are required as primary sources of carbon and nitrogen. Glucose could not be replaced by various organic acids or amino acids. Of several sugars tested, fructose, mannose, cellobiose, trehalose, maltose (weak), glycerol (weak), and the amino sugars glucosamine, N-acetylglucosamine, and N-acetylmuramic acid supported growth in the absence of glucose. Evidence was found that chitin and cell walls of starter bacteria (Lactococcus lactis) supported survival of L. monocytogenes, which suggests that the pathogen may obtain carbon and energy sources during colonization of some foods, such as cheeses, by assimilating bacteria or molds that are present.     

青霉PT95菌固态发酵产生类胡萝卜素的研究

本文对青霉Ｐｅｎｉｃｉｌｌｉｕｍｓｐ．ＰＴ９５菌株在固态发酵条件下菌核内产生类胡萝卜素进行了初步研究。结果表明,在３种固态发酵培养基中,玉米粉培养基（ＳＭＡ）比麸皮２基和棉籽壳培养基更适合于ＰＴ９５菌株固态发酵产生类胡萝卜素。为了增加菌核干重和提高类胡萝卜素产率,ＳＭＡ中需要添加氮源、碳源和植物油。在所度的各种氮、碳源中,以硝酸钠和麦芽糖效果最佳。通过我试验确定了在培养基盐溶液中添加硝酸钠３ｇ／Ｌ     

重组Bacillus subtilis产B.stearothermophilus环糊精葡萄糖基转移酶的发酵优化

利用本研究室已构建的重组菌Bacillus subtilis/pBSMuL3-α/β-CGTase对产B.stearothermophilus环糊精葡萄糖基转移酶的发酵产酶进行了优化,考察了培养基中重要成分:碳源、有机氮源、无机氮源、有机与无机氮源质量比、碳源与氮质量比、金属离子种类等单因素对该重组菌产α/β-CGTase的影响,并采用正交实验对发酵培养基进行优化,对优化结果分析可知,重组菌B.subtilis/pBSMuL3-α/β-CGTase发酵产α/β-CGTase的最优培养基成本为:葡萄糖5 g/L,氮源(鱼骨蛋白胨∶NH4Cl=3∶1)25 g/L,1 mmol/L Mg^2+。在最优条件下发酵培养,α/β-CGTase的酶活由原来TB发酵培养基的9.20 U/mL提高至20.32 U/mL,是优化前酶活的2.2倍,为α/β-环糊精葡萄糖基转移酶的工业应用提供了理论支持。     

Effects of the principal nutrients on lovastatin production by Monascus pilosus

Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose-glycerol-peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.     

一株海洋好氧反硝化细菌的鉴定及其好氧反硝化特性

【目的】从处理海洋养殖循环水的生物滤器生物膜中分离到1株具有好氧反硝化活性的细菌(菌株2-8),并进一步研究了该菌的分类地位及反硝化特性。【方法】采用16S rRNA基因序列分析对菌株进行初步鉴定,采用好氧培养技术,探讨了碳源种类、起始pH、NaCl浓度、C/N、温度和摇床转速对菌株2-8好氧反硝化活性的影响。【结果】该菌株的16S rRNA基因序列与Pseudomonas segetis FR1439T(AY770691)的相似性最高,达到99.9%,因此初步鉴定菌株2-8属于假单胞菌属(Pseudomonas sp.2-8)。碳源类型和C/N对其好氧反硝化作用的影响最为显著,以柠檬酸钠为唯一碳源,C/N为15时脱氮效率最高,低C/N导致亚硝酸盐的积累;其好氧反硝化的最适温度和pH分别为30℃和7.5;菌株2-8在摇床转速为160r/min下脱氮效果最好;NaCl浓度对其反硝化活性的影响不明显。【结论】在初始硝酸氮浓度为140mg/L,以柠檬酸钠为唯一碳源、C/N为15、pH为7.5、NaCl浓度为30g/L,30℃以及160r/min摇床培养的条件下,菌株2-8在48h内脱氮率可达92%且无亚硝酸盐积累。     

Intracellular superoxide dismutase,catalase, and glutathione peroxidase activities and membrane lipid peroxide levels in Fusarium acuminatum upon environmental changes in a defined medium

The variations of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities and lipid peroxide (LPO) levels in Fusarium acuminatum, an aerobic filamentous fungus, were investigated depending on the carbon and nitrogen sources during the incubation period. Fungus was cultivated in growing medium containing either maltose or saccharose in 5-25 g/L concentration range as a carbon source and either glycine or peptone in 5-35 g/L concentration range as a nitrogen source at 28 degrees C and 100 rpm. The observed highest SOD, CAT, and GSH-Px activities were 31.2+/-0.655, 62.5+/-5.23, and 1.52+/-0.0122 IU/mg in the presence of 20 g/L maltose and 73.96+/-1.48, 74.46+/-2.94, 3.48+/-0.083 IU/mg in the 15 g/L glycine-containing medium at 16 days, respectively. At the same time, the minimum LPO level was observed at 20 g/L maltose and 15 g/L glycine compared with the other carbon and nitrogen sources. The results showed a negative correlation between antioxidant enzyme activities and membrane LPO levels in F. acuminatum cells.     

Toluene mineralization by denitrification in an up flow anaerobic sludge blanket (UASB) reactor

In order to examine the effect of easily degradable substrate such as acetate on toluene mineralization by denitrification, an upflow anaerobic sludge blanket (UASB) reactor in steady state was set up. The experimentation was carried out in two stages. Initially, the reactor was fed with a carbon loading rate of 250 mg acetate-C L-1 d-1 as electron source. Nitrate loading rate (mg ) was adjusted to obtain a constant C/N ratio of 1.4. In the second stage, five toluene-C loading rates (TLR, mg toluene-C L-1 d-1), 25, 50, 75, 100 and 125, were assessed while total carbon loading rate and C/N were maintained constant at 250 mg C L-1 d-1 and 1.4, respectively. In so doing, acetate-C loading rate (mg acetate-C L-1 d-1) was gradually substituted by toluene-C. When acetate-C was the only electron source a dissimilative denitrifying process resulted as indicated by bicarbonate yield YHCO3, mg produced/mg carbon consumed) of 0.74 +/- 0.005 and denitrifying yield (YN2, mg N2 produced/mg consumed) of 0.89 +/- 0.042. The addition of different TLR did not affect the biological process as consumption carbon efficiency (CCE) values remained up to 95% +/- 3.5 and YHCO3 and YN2 values were higher than 0.71 +/- 0.03 and 0.88 +/- 0.01, respectively. Toluene mineralization by denitrification in continuous culture was successfully achieved. A simple UASB denitrifying reactor system has promising applications for complete conversion of nitrate, toluene and acetate into N2 and CO2 with a minimal sludge production.     

Nitrogen removal from wastewaters at low C/N ratios with ammonium and acetate as electron donors

A denitrifying upflow anaerobic sludge blanket (UASB) reactor was operated at different nitrate loading rates at a C/N ratio of 1.2, with acetate as an electron donor. This resulted in an increase in the accumulation of nitrite. After this, the UASB reactor was supplemented with 100 mg NH4+-Nl(-1) d(-1), while acetate was gradually limited in the medium. This prevented nitrite accumulation at a C/N ratio of 0.6 due to an enhanced nitrite reduction rate achieved in the reactor. An increasing amount of ammonium was consumed when the C/N ratio was lowered in the medium. This suggested that ammonium was used as an alternative electron donor during denitrification, which is supported by nitrogen balances. Nitrite was shown to be toxic for the nitrogen removal process at 200-400 mg NO2--N(l(-1) when the C/N ratio was decreased to 0.4 leading to formation of ammonium. The present study showed that addition of ammonium as an alternative electron donor for denitrification achieved a nitrogen removal process with negligible accumulation of undesirable intermediates.     

Effect of various sources of organic carbon and high nitrite and nitrate concentrations on the selection of denitrifying bacteria. I. Stationary cultures

The effect of methanol, ethanol, acetic acid and glucose together with NaNO2 or KNO3 (1,000 mg N/l) on the intensity of denitrification and selection of denitrifying bacteria from the bottom sludge of nitrogenous wastewater reservoir was examined. Denitrification was found to be the most efficient in medium with ethanol or acetic acid. The presence of glucose facilitated the selection of Alcaligenes faecalis whereas the other carbon sources enabled the selection of bacteria of the genus Pseudomonas: methanol -- P. fluorescens, ethanol -- P. mendocina. In medium with acetic acid species selection depends on the form of nitrogen: NaNO2 -- P. fluorescens, KNO3 -- P. aeruginosa.     

Utilization of Organic Nitrogen Sources by Two Phytoplankton Species and a Bacterial Isolate in Pure and Mixed Cultures

Algal production of dissolved organic carbon and the regeneration of nutrients from dissolved organic carbon by bacteria are important aspects of nutrient cycling in the sea, especially when inorganic nitrogen is limiting. Dissolved free amino acids are a major carbon source for bacteria and can be used by phytoplankton as a nitrogen source. We examined the interactions between the phytoplankton species Emiliania huxleyi and Thalassiosira pseudonana and a bacterial isolate from the North Sea. The organisms were cultured with eight different amino acids and a protein as the only nitrogen sources, in pure and mixed cultures. Of the two algae, only E. huxleyi was able to grow on amino acids. The bacterium MD1 used all substrates supplied, except serine. During growth of MD1 in pure culture, ammonium accumulated in the medium. Contrary to the expectation, the percentage of ammonium regenerated from the amino acids taken up showed no correlation with the substrate C/N ratio. In mixed culture, the algae grew well in those cultures in which the bacteria grew well. The bacterial yields (cell number) were also higher in mixed culture than in pure culture. In the cultures of MD1 and T. pseudonana, the increase in bacterial yield (number of cells) over that of the pure culture was comparable to the bacterial yield in mixed culture on a mineral medium. This result suggests that T. pseudonana excreted a more-or-less-constant amount of carbon. The bacterial yields in mixed cultures with E. huxleyi showed a smaller and less consistent difference than those of the pure cultures of MD1. It is possible that the ability of E. huxleyi to use amino acids influenced the bacterial yield. The results suggest that interactions between algae and bacteria influence the regeneration of nitrogen from organic carbon and that this influence differs from one species to another.     

Defined medium for Aquaspirillum serpens VHL effective in batch and continuous culture.

A defined medium for Aquaspirillum serpens VHL allows the replacement of the complex media now in use. It was developed by batch culture methods but supports growth in continuous culture. A basal salts medium supplemented with L-aspartic acid, L-alanine, and L-glutamic acid provided the best growth (turbidity), as long as ammonium chloride was omitted. Ammonium chloride caused either a lag or a reduction or a complete inhibition of the growth of A. serpens VHL on the above amino acids and other organic supplements depending on the combination used. Ammonium sulfate and ammonium hydroxide with L-glutamic acid allowed growth, but the lag period was increased in shake flask cultures. Vitamins, cysteine hydrochloride, and carbon dioxide had no effect on the growth rate. Viability (less than 50%) was inadequate to maintain continuous culture with L-glutamic acid as the sole source of carbon and nitrogen. Combinations of amino and carboxylic acids were then tested and, of these, L-glutamic acid (1 g/liter) and L-histidine (75 mg/liter) without ammonium chloride in the basal salts medium supported growth in batch and continuous culture. L-Glutamic acid was the limiting substrate for growth.     

Formation of Short-Chain Fatty Acids from H(2) and CO(2) by a Mixed Culture of Bacteria

The biological utilization of CO(2) and H(2) for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37 degrees C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.     

Effect of nitrates on biotransformation of phosphogypsum and phenol uptake in cultures of autochthonous sludge microflora from petroleum refining wastewaters

The effect of nitrates on the biotransformation of phosphogypsum at 30 degrees C in stationary cultures of anaerobic, heterogeneous microflora growing in medium with phenol (250-1,000 mg/L) as sole carbon source was studied. The microorganisms used in this study were isolated from sludge in biological petroleum-refining wastewater treatment plant. Phosphogypsum (a waste product in the chemical industry that contains approximately 95% CaSO4) was added in amount of 5 g/L, the source of nitrates was KNO3 in concentration equivalent to that of phenol (250-1,000 mg N-NO3/L). The presence of nitrates in heterogeneous cultures has an inhibitory effect on the process of phosphogypsum biotransformation and stimulates the uptake of phenol. We have found that in cultures in medium containing phenol, phosphogypsum and nitrates at least three physiological groups of microorganisms were present. These were phenol-biodegrading microorganisms not requiring an external electron acceptor, sulfate-reducing bacteria biodegrading phenol or intermediate products of its breakdown and denitrifying bacteria not utilising phenol as a carbon source. On solid medium these bacteria together formed heterogeneous single colonies. In spite of repeated attempts we were unable to isolate pure strains and the only result of these measures was loss of denitrification ability in medium with phenol.     

Isolation and characterization of a Pseudomonas putida strain able to grow with trimethyl-1,2-dihydroxy-propyl-ammonium as sole source of carbon, energy and nitrogen

Trimethyl-1,2-dihydroxypropyl-ammonium (TM) originates from the hydrolysis of the parent esterquat surfactant, which is widely used as softener in fabric care. Based on test procedures mimicking complex biological systems, TM is supposed to degrade completely when reaching the environment. However, no organisms able to degrade TM were isolated nor has the degradation pathway been elucidated so far. We isolated a Gram-negative rod able to grow with TM as sole source of carbon, energy and nitrogen. The strain reached a maximum specific growth rate of 0.4(h-1) when growing with TM as the sole source of carbon, energy and nitrogen. TM was degraded to completion and surplus nitrogen was excreted as ammonium into the growth medium. A high percentage of the carbon in TM (68% in continuous culture and 60% in batch culture) was combusted to CO2 resulting in a low yield of 0.54 mg cell dry weight per mg carbon during continuous cultivation and 0.73 mg cell dry weight per mg carbon in batch cultures. Choline, a natural structurally related compound, served as a growth substrate, whereas a couple of similar other quaternary aminoalcohols also used in softeners did not. The isolated bacterium was identified by 165-rDNA sequencing as a strain of Pseudomonas putida with a difference of only one base pair to P. putida DSM 291T. Despite their high identity, the reference strain P. putida DSM 291T was not able to grow with TM and the two strains differed even in shape when growing on the same medium. This is the first microbial isolate able to degrade a quaternary ammonium softener head group to completion. Previously described strains growing on quaternary ammonium surfactants (decyltrimethylammonium, hexadecyltrimethylammonium and didecyldimethylammonium) either excreted metabolites or a consortium of bacteria was required for complete degradation.     

Optimization of lipid production in the oleaginous yeastApiotrichum curvatum in wheypermeate

Summary Lipid production of the oleaginous yeastApiotrichum curvatum was studied in wheypermeate to determine optimum operation conditions in this medium. Studies on the influence of the carbon to nitrogen ratio (C/N-ratio) of the growth medium on lipid production in continuous cultures demonstrated that cellular lipid content in wheypermeate remained constant at 22% of the cell dry weight up to a C/N-ratio of about 25. The maximal dilution rate at which all lactose is consumed in wheypermeate with excess nitrogen was found to be 0.073 h^-1. At C/N-ratios higher than 25–30 lipid content gradually increased to nearly 50% at C/N=70 and the maximal obtainable dilution rate decreased to 0.02 h^-1 at C/N=70. From these studies it could be derived that maximal lipid production rates can be obtained at C/N-ratios of 30–35 in wheypermeate. Since the C/N-ratio of wheypermeate normally has a value between 70 and 101, some additional nitrogen is required to optimize the lipid production rate. Lipid production rates ofA. curvatum in wheypermeate were compared in four different culture modes: batch, fed-batch, continuous and partial recycling cultures. Highest lipid production rates were achieved in culture modes with high cell densities. A lipid production rate of nearly 1 g/l/h was reached in a partial recycling culture. It was calculated that by using this cultivation technique lipid production rates of even 2.9 g/l/h may be reached when the supply of oxygen can be optimized.Nomenclature C/N-ratio carbon to nitrogen ratio of the growth medium (g/g) - C/N_crit C/N-ratio at which there is just enough nitrogen to allow all carbon source to be converted to biomass - D dilution rate=volume of incoming medium per unit time/volume of medium in the culture vessel (h^-1) - D_max maximum dilution rate (h^-1) - DW cell dry weight - L lipid yield (g storage lipid/g carbon source) - specific growth rate (h^-1) - _max maximum specific growth rate (h^-1) - Q_L lipid production rate (g/l/h) - Y_i molecular fraction of carbon substrate that is converted to storage carbohydrate (C-mol/C-mol) - Y_ls maximal amount of storage lipid that can be produced per mol carbon source (C-mol/C-mol)     

A Pressurized Chemostat for the Study of Marine Barophilic and Oligotrophic Bacteria

A continuous culture system that allows bacteria to be grown in steady-state populations under pressures of up to 700 atm (71 MPa) was constructed and tested. With readily available or slightly modified high-pressure chromatography equipment, a continuous flow of sterile medium is pressurized and passed through a 500-ml nylon-coated titanium reactor at flow rates of 0.01 to 10 ml min(sup-1). The pressure in the reactor is controlled by a backpressure regulator with greater than 1% accuracy. In test experiments, a culture of a psychro- and barophilic marine isolate from a depth of 4,900 m (strain F1-A, identified as a Shewanella sp.) was grown at 1, 300, and 450 atm (0.1, 30.4, and 40.5 MPa) and dilution rates of 60 and 90% of the organism's maximum growth rate (determined at 1 atm) in the required complex medium at levels of 3.3 and 0.33 mg of dissolved organic carbon per liter in the reservoir. Growth limitation by carbon was assured by an appropriate C/N/P ratio of the medium. The data indicate that barophilic growth characteristics in steady-state cultures of this psychro- and barophilic deep-sea isolate were positively affected by a decreasing growth rate at the higher of two substrate concentrations in the reservoir. After a 10-fold lowering of the substrate concentration, the effect was reversed. Under these conditions, the cell viability increased significantly, especially at the higher of the two pressures tested. The basic design of the system can principally also be used for growth studies on hyperthermophilic bacteria and archaea.     

Modelling interactions between phytoplankton and bacteria under nutrient-regenerating conditions

The interactions of phytoplankton and bacteria in a nitrogen-limitedsteady-state system with an organic nitrogen compound or ammoniumas the sole nitrogen source were modelled. The effects of variousalgal excretion rates and two different mathematical representationsof excretion were examined. The model predicted that higherexcretion elevated the bacterial steady-state biomass, and loweredthe algal biomass. Bacterial respiration, which directly determinednitrogen regeneration, had an important effect on the system.The bacterial growth yield in the model was mainly a functionof the growth rate, and not of the nitrogen:carbon ratio ofthe substrate. In one version of the model, where the excretionof organic carbon increased with decreasing growth rate, themodel started to oscillate when the multiplication product ofmaximum specific excretion of excreted organic carbon (EOC)and the bacterial yield on EOC exceeded the dilution rate, irrespectiveof the form of nitrogen (ammonium or dissolved organic nitrogen)in the medium. The model results were compared with chemostatexperiments with the alga Emiliania huxleyi and a bacterialisolate in pure and mixed culture at two different dilutionrates. The carbon and nitrogen biomass of the bacteria was {smalltilde}1.5 times higher in mixed culture than in pure culture.In the experiments with low dilution rate, the recovery of nitrogenin the form of biomass, ammonium or amino acids was low, suggestingthe excretion by the algae of a refractory nitrogen-containingproduct which the bacteria could not use.