Modeling of recycling oxic and anoxic treatment system for swine wastewater using neural networks

A recycling reactor system operated under sequential anoxic and oxic conditions for the treatment of swine wastewater has been developed, in which piggery slurry is fermentatively and aerobically treated and then part of the effluent is recycled to the pigsty. This system significantly removes offensive smells (at both the pigsty and the treatment plant), BOD and others, and may be cost effective for small-scale farms. The most dominant heterotrophic were, in order,Alcaligenes faecalis, Brevundimonas diminuta andStreptococcus sp., while lactic acid bacteria were dominantly observed in the anoxic tank. We propose a novel monitoring system for a recycling piggery slurry treatment system through the use of neural networks. In this study, we tried to model the treatment process for each tank in the system (influent, fermentation, aeration, first sedimentation and fourth sedimentation tanks) based upon the population densities of the heterotrophic and lactic acid bacteria. Principal component analysis (PCA) was first applied to identify a relationship between input and output. The input would be microbial densities and the treatment parameters, such as population densities of heterotrophic and lactic acid bacteria, suspended solids (SS), COD, NH₄ ⁺-N, or-tho-phosphorus (o-P), and total-phosphorus (T-P). Then multi-layer neural networks were employed to model the treatment process for each tank. PCA filtration of the input data as microbial densities was found to facilitate the modeling procedure for the system monitoring even with a relatively lower number of input. Neural networks independently trained for each treatment tank and their subsequent combined data analysis allowed a successful prediction of the treatment system for at least two days.     

Hybrid neural network modeling of a full-scale industrial wastewater treatment process

In recent years, hybrid neural network approaches, which combine mechanistic and neural network models, have received considerable attention. These approaches are potentially very efficient for obtaining more accurate predictions of process dynamics by combining mechanistic and neural network models in such a way that the neural network model properly accounts for unknown and nonlinear parts of the mechanistic model. In this work, a full-scale coke-plant wastewater treatment process was chosen as a model system. Initially, a process data analysis was performed on the actual operational data by using principal component analysis. Next, a simplified mechanistic model and a neural network model were developed based on the specific process knowledge and the operational data of the coke-plant wastewater treatment process, respectively. Finally, the neural network was incorporated into the mechanistic model in both parallel and serial configurations. Simulation results showed that the parallel hybrid modeling approach achieved much more accurate predictions with good extrapolation properties as compared with the other modeling approaches even in the case of process upset caused by, for example, shock loading of toxic compounds. These results indicate that the parallel hybrid neural modeling approach is a useful tool for accurate and cost-effective modeling of biochemical processes, in the absence of other reasonably accurate process models.     

Evaluation of spectrofluorometry as a tool for estimation in fed-batch fermentations

Native culture fluorescence was investigated as an additional source of information for predicting biomass and glucose concentrations in a fed-batch fermentation of Alcaligenes eutrophus. Partial least squares (PLS) regression and a feed forward neural network (FFNN) coupled with principle component analysis (PCA) were each used to model the kinetics of the fermentation. Data from three fermentations was combined to form a training set for model calibration and data from a fourth fermentation was used as the testing set. The fluorescent soft-sensors were compared with a previously developed feed forward neural network soft-sensor model which used oxygen uptake rate (OUR), carbon dioxide evolution rate (CER), aeration rate, feed rate, and fermentor volume to estimate biomass and glucose concentrations. The best model performance for predicting both biomass and glucose concentrations was achieved using the native fluorescence-based models. Real data predictions of the biomass concentration in the testing set were obtained using both the PLS and FFNN PCA modeling utilizing fluorescence measurements plus the rate of change of the fluorescence measurements. Accurate predictions of the glucose concentration in the testing set were obtained using the FFNN PCA modeling technique utilizing the rate of change of the fluorescence measurements. Substrate exhaustion was indicated qualitatively by a first-order PLS model utilizing the rate of change of fluorescence measurements. These results indicate that native culture fluorescence shows promise for providing additional valuable information to enhance predictive modeling which cannot be extracted from other easily acquired measurements.     

云锦杜鹃不同花期挥发性成分的HS-SPME-GC-MS检测与主成分分析

为了探究云锦杜鹃的挥发性成分,该研究采用顶空固相微萃取-气相色谱-质谱联用技术对云锦杜鹃不同花期中的挥发性成分进行定性定量分析,并通过主成分分析法分析其特征挥发性成分。结果表明:四个花期共检测出50种挥发性成分,共分为苯丙酸类/苯环型、萜烯类、醇类、醛类、烃类和其他类六大类组分。对29种主要挥发性物质进行主成分分析,提取了两个主成分,累计方差贡献率达到88.545%。分析发现,β-月桂烯、β-罗勒烯、可巴烯、异喇叭烯、桉树脑、衣兰烯、(+)-表二环倍半水芹烯、(3R-反式)-4-乙烯基-4-甲基-3-(1-甲基乙烯基)-1-(1-甲基乙基)-环己烯与第1主成分呈高度正相关,第2主成分的贡献率为31.455%,其中丁香酚的影响最大,呈高度负相关,这些物质是影响云锦杜鹃香气的关键性成分。在9种高度相关的物质中,萜烯类物质占了7种。综上认为,萜烯类物质是云锦杜鹃主要特征香气成分。     

Fed batch bioconversion of 2-propanol by a solvent tolerant strain of <Emphasis Type="Italic">Alcaligenes </Emphasis><Emphasis Type="Italic">faecalis</Emphasis> entrapped in Ca-alginate gel

A gram-negative, rod-shaped, aerobe, capable of converting 2-propanol (isopropanol, IPA) to acetone was isolated from an oil/sump, and identified by 16 S rDNA analysis as Alcaligenes faecalis. Investigations showed this strain to be extremely solvent-tolerant and it was subsequently named ST1. In this study, A. faecalis ST1 cells were immobilized by entrapment in Ca-alginate beads (3 mm in diameter), and used in the bioconversion of high concentration IPA. The biodegradation rates and the corresponding microbial growth inside the beads were measured at four different IPA concentration ranges from 2 to 15 g l(-1). The maximum cell concentration obtained was 9.59 g dry cell weight (DCW) l(-1) medium which equated to 66 g DCW l(-1) gel, at an initial IPA concentration of 15 g l(-1) after 216 h of incubation. A maximum biodegradation rate of 0.067 g IPA g cells(-1) h(-1) was achieved for 5 g l(-1) IPA where an increase in IPA concentration to 38 g l(-1) caused reduction in bead integrity. A modified growth medium was developed which allowed repeated use of the beads for more than 42 days without any loss of integrity and continued bioconversion activity.     

Novel Leuconostoc citreum starter culture system for the fermentation of kimchi, a fermented cabbage product

To determine the dominant microorganisms involved in kimchi fermentation and to examine their effect on kimchi fermentation, we randomly isolated and characterized 120 lactic acid bacteria from kimchi during a 5-day fermentation at 15°C. Leuconostoc citreum was dominant during the early and mid-phases of kimchi fermentation whereas Lactobacillus sake/Lactobacillus curvatus or Lactobacillus brevis were found during later stages. Eighty-two out of 120 isolates (68%) were identified as Leuconostoc citreum by means of a polyphasic method, including 16S rDNA sequencing and DNA/DNA hybridization. A few Weissella confusa-like strains were also isolated during the mid-phase of the fermentation. Strain IH22, one of the Leuconostoc citreum isolates from kimchi, was used as an additive to evaluate growth and acid production in kimchi fermentation. This strain was consistently over 95% of the population in IH22-treated kimchi over a 5-day fermentation, while heterogeneous lactic acid bacteria were observed in the control kimchi. The pH in IH22-treated kimchi dropped rapidly but was stably maintained for 5 days, compared to its slow and prolonged decrease in the control kimchi. These results indicate that Leuconostoc citreum IH22 dominates over and retards the growth of other lactic acid bacteria in kimchi, suggesting it can be used as a bacterial starter culture to maintain the quality of kimchi for prolonged periods. This revised version was published online in June 2006 with corrections to the Cover Date.     

Model systems for the immunolocalisation of cis,trans abscisic acid in plant tissues

To explore the feasibility of immunolocalisation of endogenous abscisic acid (ABA), model systems were developed for testing quantitatively the sensitivity of the second antibody peroxidase/antiperoxidse (PAP) method for immunolocalisation of ABA on plant tissues. Exogenous (±)ABA was fixed to carrot sections on glass slides or to homogenised pea cotyledon material on microtitre plates, either directly by carbodiimide fixation or by glutaraldehyde fixation of ABA-protein conjugates linked through the C1 carboxyl by 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride (EDC). Backgrounds were decreased by including 0.1% normal goat serum in the incubations, by including 0.1% Triton X-100 as a wetter, by including glycine in the rinses after EDC fixation and by using low-pH rinses after incubation with the primary antibody. Serum antibodies recognising the peptide bond between the protein and abscisic acid were removed by preincubating the serum with acetic acid conjugated to protein. Positives were only accepted when they could be eliminated by adding an excess of ABA-protein conjugate in the primary antiserum. By using a soluble peroxidase reaction product to facilitate quantitation, the limit of reliable exogenous ABA detection was found to be only of the order of 1 pmol. For the histochemical immunolocalisation of endogenous ABA, better antisera and lower backgrounds will be required.The efficiency of fixation of exogenous ABA was determined using [³H] or [¹⁴C]ABA. When aqueous EDC or di-isopropyl carbodiimide (IPC) were used the fixation efficiency was low (up to 5%), but much higher efficiencies (up to 80%) were obtained using IPC vapour with freeze-dried material. Similarly efficient fixation of endogenous ABA in pea cotyledon material, as determined by gas chromatography-mass spectrometry analysis, was obtained using the same technique. The PAP method failed to detect fixed endogenous ABA in pea cotyledons, even though the total tissue amounts present exceeded 1 pmol, evidence that not enough of the ABA was accessible to the antibody.Abbreviations ABA abscisic acid - ACE-ALP acetic acid-alkaline phosphatase - EDC 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride - GC-MS gas chromatographymass spectrometry - IgG Immunoglobulin G - HSA humanserum albumin - IPC dinsopropyl carbodiimide - LINK goat anti-rabbit IgG - OD optical density - PAP peroxidase/rabbit antiperoxidase complex     

ent-Kaurene biosynthesis in a cell-free system from wheat (Triticum aestivum L.) seedlings and the localisation of ent-kaurene synthetase in plastids of three species

A cell-free system capable of converting [¹⁴C]geranylgeranyl diphosphate to ent-[¹⁴C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [¹⁴C]geranylgeranyl diphosphate into ent-[¹⁴C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[¹⁴C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.Abbreviations ADH alcohol dehydrogenase - AMO 1618 2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate - BSA bovine serum albumin - DTT dithioth-reitol - GA_n gibberellin A_n - GAPDH NADP⁺-glyceraldehyde 3-phosphate dehydrogenase - GC-MS combined gas chromatography-mass spectrometry - GGPP all trans-isomer of geranyl-geranyl diphosphate - KS ent-kaurene synthetase - MDH malate dehydrogenase - MAA mevalonate activating activity - SOR shikimate oxidoreductase We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[²H₂]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft.     

Isolation and characterization of a bacterium that degrades various polyester-based biodegradable plastics

Microorganisms isolated from soil samples were screened for their ability to degrade various biodegradable polyester-based plastics. The most active strain, designated as strain TB-13, was selected as the best strain for degrading these plastics. From its phenotypic and genetic characteristics, strain TB-13 was closely related to Paenibacillus amylolyticus. It could degrade poly(lactic acid), poly(butylene succinate), poly(butylene succinate-co-adipate), poly(caprolactone) and poly(ethylene succinate) but not poly(hydroxybutylate-co-valerate). However, it could not utilize these plastics as sole carbon sources. Both protease and esterase activities, which may be involved in the degradation of plastic, were constitutively detected in the culture broth.     

Secretory granule formation and membrane recycling by the trans-Golgi network in adipokinetic cells of Locusta migratoria in relation to flight and rest

The influence of flight activity on the formation of secretory granules and the concomitant membrane recycling by the rans-Golgi network in the peptidergic neurosecretory adipokinetic cells of Locusta migratoria was investigated by means of ultrastructural morphometric methods. The patterns of labelling of the trans-Golgi network by the exogenous adsorptive endocytotic tracer wheat-germ agglutinin-conjugated horseradish peroxidase and by the endogenous marker enzyme acid phosphatase were used as parameters and were measured by an automatic image analysis system. The results show that endocytosed fragments of plasma membrane with bound peroxidase label were transported to the trans-Golgi network and used to build new secretory granules. The amounts of peroxidase and especially of acid phosphatase within the trans-Golgi network showed a strong tendency to be smaller in flight-stimulated cells than in non-stimulated cells. The amounts of acid phosphatase in the immature secretory granules originating from the trans-Golgi network were significantly smaller in stimulated cells. The number of immature secretory granules positive for acid phosphatase tended to be higher in stimulated cells. Thus, flight stimulation of adipokinetic cells for 1 h influences the functioning of the trans-Golgi network; this most probably results in a slight enhancement of the production of secretory granules by the trans-Golgi network.     

Diversity and dynamics of bacterial species in a biofilm at the end of the Seoul water distribution system

To investigate changes in the bacterial species and hygienic safety of the biofilm at the end of the drinking water distribution system in Seoul (Korea), denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to analyse the bacterial population in the biofilm of a semi-pilot galvanized iron pipe model. The presence of sequences from aerobic Sphingomonas sp., anaerobic Rhodobacter sp., and unculturable bacteria indicated that these organisms coexisted after 1 day of model operation, demonstrating the ease of biofilm formation on galvanized iron pipes in the end region of the water distribution system studied. Sequences similar to those of unculturable bacteria, E. coli, and anaerobic bacteria were detected during the course of succession on the biofilm. More complicated band patterns were observed after 70 days of operation. PCR-DGGE illustrated changes in the biofilm during succession as well as the possibilities of anaerobic conditions and faecal contamination of the drinking water system. PCR-DGGE and culture-dependent fatty acid methyl ester (FAME) analysis showed different patterns for the same samples (Lee & Kim 2003); however, PCR-DGGE showed less diversity than did FAME analysis. This study compared the culture-dependent FAME and culture-independent PCR-DGGE methods directly, and their use in promoting the hygienic safety of drinking water.     

Metabolism of gibberellins in a cell-free system from immature seeds of Phaseolus vulgaris L.

The biosynthetic steps from gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) to C₁₉-GAs were studied by means of a cell-free system from the embryos of immature Phaseolus vulgaris seeds. Stable-isotope-labeled GAs were used as substrates and the products were identified by gas chromatography-mass spectrometry. Gibberellin A₁₂-aldehyde was converted to GA₄ via non-hydroxylated intermediates and to GA₁ via 13-hydroxylated intermediates. 13-Hydroxylation took place at the beginning of the pathway by the conversion of GA₁₂-aldehyde to GA₅₃-aldehyde. The conversion of GA₂₀ to GA₅ and GA₆ was also shown but no 2-hydroxylating activity was found. Endogenous GAs from embryos and testas of 17-dold seeds were re-examined by gas chromatography-selected ion monitoring using stable-isotopelabeled GAs as internal standards. Gibberellins A₉, A₁₂, A₁₅, A₁₉, A₂₃, A₂₄, and A₅₃ were identified for the first time in P. vulgaris, in addition to GA₁, GA₄, GA₅, GA₆, GA₈, GA₁₇, GA₂₀, GA₂₉, GA₃₇, GA₃₈ and GA₄₄, which were previously known to occur in this species. The levels of all GAs, except the 2-hydroxylated ones, were greater in the embryos than in the testas. Conversely, the contents of GA₈ and GA₂₉, both 2-hydroxylated, were much higher in the testas than in the embryos.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - GC-SIM gas chromatography-selected ion monitoring - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - m/z ion of mass     

ICP-MS法测定广西金樱根及炮制品中22种金属元素

为建立广西金樱根药材中22种金属元素的电感耦合等离子体质谱(ICP-MS)分析方法,该文对不同产地药材及其炮制品中金属元素含量进行比较分析,评价其药材的质量和优选适当的炮制方法.同时,采用ICP-MS法测定22种金属元素含量,绘制其特征金属元素柱状图谱,并采用系统聚类分析和主成分分析对金樱根药材不同产地、不同炮制品的4...     

New evidence for a role for thioredoxin h in germination and seedling development

Thioredoxin of the h-type — earlier linked to the reduction of wheat (Triticum durum Desf. cv. Monroe) endosperm proteins — was converted from an oxidized to a partially reduced state during germination and seedling development. While the abundance of thioredoxin progressively decreased during this period, the availability of reducing equivalents, defined as the product of the relative abundance of thioredoxin and the percent reduction, increased. The amount of the enzyme catalyzing the reduction of thioredoxin h (NADP-thioredoxin reductase) remained constant. The activities of enzymes generating the NADPH needed for the reduction of thioredoxin (glucose 6-phosphate and 6-phosphogluconate dehydrogenases) increased. The level of thioredoxin h in the endosperm appeared to be controlled by the embryo via hormones. Gibberellic acid enhanced the disappearance of thioredoxin, whereas abscisic acid showed the opposite effect. Moreover, uniconazole, an inhibitor of gibberellic acid synthesis, slowed seedling growth and inhibited the disappearance of thioredoxin in a manner reversible by gibberellic acid. The results are consistent with a role for thioredoxin h in initiating the mobilization of nitrogen and carbon needed for germination and seedling development.Abbreviations ABA cis-abscisic acid - DTT dithiothreitol - GA gibberellin - GA₃ gibberellic acid - mBBr monobromobimane - NTR NADP-thioredoxin reductase This work was supported by National Science Foundation grant MCB-9316496. We thank Dr. Yuji Kamiya for advice and the Sumitomo Chemical Co. for a generous sample of uniconazole.     

Chiral separation of some 4a-methyl-1,2,3,4,4a,9a-hexahydro-fluoren-9-one derivatives as a probe for difference in solvation by 2-propanol of carbamate moiety in chiralcel OD-H,chiralpak AD,and chiralpak AS chiral stationary phases

Chromatographic resolution of 12 derivatives in the 4a-methyl-2,3,4,4a-tetrahydro-1H-fluorene and 4a-methyl-1,2,3,4,4a,9a-hexahydrofluoren-9-one series differing by the framework around position 9 and substitution in position 6, are reported on Chiralcel OD, Chiralpak AD, and Chiralpak AS under two elution conditions and according to the two classes of enantiomers. Results from principal component analysis (PCA) as well as hierarchical clustering show a clustering of the actual compounds depending on properties around position 9, the effect of the substituent in position 6 (methyl, chloro or fluoro) not being strong enough to intermesh the data. Carbamate phases show very different properties when they are used in the separation of a series of ketones C and α-chloroketones D , which differ in basicity and the steric requirement around the carbonyl. Analysis of the effect of 2-PrOH content in hexane on the retention is consistent with a large difference in solvation of the carbamate moiety by 2-PrOH, in the order Chiralcel OD > Chiralpak AD > Chiralpak AS. 4a-Methyl-2,3,4,4a-tetrahydro-1H-fluorene derivatives, which lack hydrogen bonding sites, are not discriminated on these CSPs and show identical k′ responses to 2-PrOH content changes on the three CSPs. Chirality 10:770–777, 1998. © 1998 Wiley-Liss, Inc.     

Validation of a radioimmunoassay for (+)-abscisic acid in extracts of apple and sweet-pepper tissue using high-pressure liquid chromatography and combined gas chromatography-mass spectrometry

A radioimmunoassay for (+)-abscisic acid (ABA) was developed and applied to the analysis of free ABA in extracts of apple (Malus pumila Mill.) and sweet pepper (Capsicum annuum L.) leaves at various stages during extract purification. Conjugates of ABA, were quantified after alkaline hydrolysis. The validity of the radioimmunoassay was tested by comparison of immunoassay estimates of ABA at different levels of extract purity with high-pressure liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry. The antiserum, raised against (+)-ABA, was almost equally sensitive to (-)-ABA. Serum cross-reactivity with the methyl ester of ABA was 160% and with the glycosyl ester of ABA was 34%. Cross-reactivity with protein-ABA conjugates was very slight for C₄-conjugated keyholelimpet haemocyanin, but about 1000% for C₁-conjugated alkaline phosphatase. Other compounds tested showed extremely low or undetectable cross-reactivities. Further evidence for the specificity of the assay came from the agreement between the results using different assay methods for both apple and pepper extracts, and from the observation that the only zone of immunoreactivity on HPLC elution profiles corresponded with authentic (+)-ABA. The use of polyvinylpyrrolidone in the assay minimised interference by other substances in plant extracts. In pepper, free ABA levels increased rapidly during water stress and recovered to pre-stress levels within two days after rewatering. Levels of ABA conjugates were significantly lowr than free ABA in unstressed plants, and also increased rapidly with stress, although not to the same extent as free ABA, and did not recover as rapidly as did free ABA. In apple, levels of free ABA and of ABA conjugates both increased more than twofold over a two-week period of water stress. In contrast to pepper, however, immunoreactivity of the conjugate fraction was increased by hydrolysis, indicating that different ABA conjugates predominate in the two species.Abbreviations ABA abscisic acid - GC-MS combined gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography - Me-ABA methyl ester of ABA - PVP polyvinylpyrrolidone - RIA radioimmunoassay     

p-Coumaric acid — a monomer in the sporopollenin skeleton

Sporopollenin obtained from wings of Pinus mugo (Turra) pollen was analysed by pyrolysis mass spectrometry. In the spectrum, mass peaks which are characteristic for p-coumaric acid were dominant. p-Coumaric acid was the main degradation compound when the wing material was treated by a gentle method using AII₃, and also when the remaining residue of the treated sporopollenin material was saponified. It is therefore assumed that p-coumaric acid is a genuine structural unit in the sporopollenin skeleton. In addition, the effects of AII₃ treatment indicate that the p-coumaric acid might be bound by ether linkages.Abbreviations HPLC high-performance liquid chromatography - MS mass spectrometry - TLC thin-layer chromatography     

Evidence for a simple Cl conductance pathway in nutrient membrane of frog stomach

A decrease in nutrient Cl⁻ results in an increased negativity of the nutrient relative to the secretory side. The possibility emerged that Cl⁻ transport could be attributed to a neutral mechanism involving Cl⁻ in the nutrient membrane coupled to a simple Cl⁻ conductance pathway in the secretory membrane. The decrease in PD (potential difference) with a decrease in nutrient Cl⁻ could arise from a decrease in cellular Cl⁻ so that the ratio of Cl⁻ in cell to Cl⁻ in secretory solution was decreased. Experiments were designed to determine whether there was a need to assume a simple Cl⁻ conductance pathway. A 10-fold decrease in Cl⁻ gave in HCO₃⁻-containing nutrient solutions a PD decrease of 20 mV, in HCO₃⁻-free nutrient solutions, a PD decrease of 13.5 mV, and in HCO₃⁻-free and Na⁺-free solutions, a PD decrease of 6.7 mV. The decrease of 6.7 mV could not be attributed to a neutral Cl⁻HCO₃⁻ exchanger or a NaCl symport. Also there was no evidence for a KCl symport from changes in Cl⁻ in presence and absence of K⁺. It followed that the decrease of 6.7 mV provided evidence for a simple Cl⁻ conductance pathway in the nutrient membrane.