Butanol Yield Research Articles

The Effect of Technological Conditions on ABE Fermentation and Butanol Production of Rye Straw and the Composition of Volatile Compounds.

The objective of this study was to evaluate the effect of pretreatment and different technological conditions on the course of ABE fermentation of rye straw (RS) and the composition of volatile compounds in the distillates obtained. The highest concentration of ABE and butanol was obtained from the fermentation of pretreated rye straw by alkaline hydrolysis followed by detoxification and enzymatic hydrolysis. After 72 h of fermentation, the maximum butanol concentration, productivity, and yield from RS were 16.11 g/L, 0.224 g/L/h, and 0.402 g/g, respectively. Three different methods to produce butanol were tested: the two-step process (SHF), the simultaneous process (SSF), and simultaneous saccharification with ABE fermentation (consolidation SHF/SSF). The SHF/SSF process observed that ABE concentration (21.28 g/L) was higher than in the SSF (20.03 g/L) and lower compared with the SHF (22.21 g/L). The effect of the detoxification process and various ABE fermentation technologies on the composition of volatile compounds formed during fermentation and distillation were analyzed.

Novel Batch and Repeated-Batch Butanol Fermentation from Sweet Sorghum Stem Juice by Co-Culture of Arthrobacter and Immobilized Clostridium in Scaled-Up Bioreactors

This research aims to study butanol fermentation from sweet sorghum stem juice (SSJ) by immobilized Clostridium beijerinckii TISTR 1461 cells on bamboo chopsticks using Arthrobacter sp. as an efficient bacterium for creating anaerobic conditions in scaled-up bioreactors. For batch culture in a 1-L screw-capped bottle, a butanol concentration (PB), butanol productivity (QB), and butanol yield (YB/S) were 12.09 g/L, 0.26 g/L·h and 0.28 g/g, respectively. These values were ~8 to 14% higher than those of a single culture using oxygen-free nitrogen (OFN) gas to generate anaerobic conditions. When butanol fermentation by the co-culture was scaled-up to 5-L and 30-L stirred-tank fermenters, the butanol production efficiency was not different from that using the 1-L bottles. Additionally, repeated-batch butanol fermentation in the 1-L bottles by the co-culture was successfully operated for four successive cycles with high butanol production. All results clearly indicate that Arthrobacter sp. is promising for creation of anaerobic conditions for butanol production by immobilized Clostridium in large scale bioreactors.

Biosynthesis of NPs CuS/Cu2S and self-assembly with C. beijerinckii for improving lignocellulosic butanol production in staged butyrate-butanol fermentation process

A staged butyrate-butanol fermentation process (SBBFP) has been developed to efficiently produce biobutanol from renewable lignocellulose. The SBBFP combines the fermentation of bioaugmented degradation consortium for butyrate (BDCB) and butanol production of Clostridium beijerinckii, but is still challenged by the lower specific butanol yield (SBY). In this study, a novel technology for synthesizing semiconductor nanoparticles (NPs) and their self-assembly with Clostridium beijerinckii was proposed to enhance the SBY. Results showed that supplement of 0.1 g/L CuSO4 or 0.2 g/L ZnSO4 were more favorable for the formation of butyrate and NPs (CuS/Cu2S or ZnS) in the acidogenic fermentation process of BDCB. Butyrate production achieved 19–21 g/L in the fed-batch fermentation mode from NaOH-pretreated rice straw. Only CuS/Cu2S can be assembled with NPs@C. beijerinckii hybrid system, and a butanol production of 14.6 g/L was obtained using acidogenic fermentation broth and saccharide liquid from rice straw as the co-substrates, with the SBY of 0.17 g/g initial pretreated rice straw. The enhanced butanol synthesis was likely attributed to the upgraded intracellular NADH/NAD+ and specific activity of enzyme CoA-transferase (ctfAB) in C. beijerinckii. The research work provides an efficient approach for biofuels and biochemical production from lignocellulose, along with the extended application of biological nanotechnology.

Mathematical modeling of ABE fermentation on glucose substrate with Zn supplementation for enhanced butanol production

Supplementation or limitation of some micronutrients during acetone-butanol-ethanol (ABE) fermentation has led to improvement in butanol yield and productivity. A mechanistic model of ABE fermentation offers insights in understanding these complex interactions and improving productivity through optimal culture conditions. This study proposes a mechanistic kinetic model of ABE fermentation by two Clostridium Acetobutylicum strains, L7 and ATCC 824 using glucose as sole carbon source without zinc and with various zinc doses. The model incorporates enzyme regulation by zinc on several glycolytic, acidogenesis and solventogenesis enzymes. The model was fitted and validated to experimental data collected from the published literature. The simulated results were in compliance with the experimental data, most importantly indicating higher glucose consumption and butanol productivity when supplemented with zinc compared to the control culture. The average squared correlation factor (R2) between the experimental and the simulated results, without and with zinc, were 0.99 and 0.96 for glucose, and 0.89 and 0.95 for butanol, respectively. A sensitivity analysis performed on the fitted and validated model indicated that the glucose consumption and growth parameters most influenced the model outputs. The developed model can be used as a template for modeling ABE fermentation under different combinations of micronutrients that may offer improved butanol yield and productivity.

Alternative Utilization of Pennisetum purpureum × Pennisetum americanum: Press Cake Conversion to Biobutanol

Conversion of Pennisetum purpureum × Pennisetum americanum (Napier Pak Chong1) press cake into biobutanol using Clostridium beijerinckii TISTR 1461 was proposed as an alternative to combustion in this study. The optimum conditions for biobutanol fermentation were determined using a full factorial design and a central composite design of experiment. The studied factors were initial pHs (5.50–6.50) and sugar concentrations (40–60 g/L), while butanol yield (g/g reducing sugar utilized) was specified as the optimization response. The results showed that the suitable enzyme loading of alkali-pretreated press cake (at 3% w/w NaOH, 10% substrate loading, boiling at 90 °C, with a reaction time of 1 h) was 10 FPU/g biomass, which provided a glucose yield of 345 mg/g pretreated press cake. The optimized pH and reducing sugar concentration were 6.08 and 43 g/L, respectively. At these conditions, the maximum butanol yield from the hydrolysate of NaOH-pretreated press cake was 0.135 g/g reducing sugar utilized (0.30 g/g glucose utilized). Apart from the possibility of generating much less pollution, it was estimated that using the same amount of press cake, butanol production could possibly have a value comparable to that obtained from combustion for electricity production. A new concept for overall Napier Pak Chong1 grass utilization was also presented.

Extractive biobutanol fermentation using immobilized cells with organic acid esters as novel co-substrates in large extractant volume

Organic acid co-substrates increase butanol production; however, their toxicity to cells limits their utilization during fermentation. In the ester form, the substrate is storable in the extractant as fermentation feedstock. This study investigated butanol fermentation by immobilized cells in a large extractant volume, using glucose and esters as co-substrates. By employing ethyl butyrate at a substantial co-substrate to glucose ratio of 1.4, an impressive total butanol of 50.1 g/L was achieved. In contrast, when utilizing acids, this ratio yielded only 10.9 g/L. Both batch and fed-batch fermentations incorporating ester feeding exhibited comparable total butanol concentrations, affirming the gradual release of esters into the medium while ensuring their concentration remained below the toxicity threshold. A high butanol yield of 0.64 was obtained using ethyl butyrate as co-substrate, reducing the sugar required to produce butanol. This result could be applied for the utilization of butyrate ester to obtain high butanol yield.

Ethanol Coupling Reactions over MgO-Al2O3 Mixed Oxide-Based Catalysts for Producing Biofuel Additives.

Catalytic conversion of ethanol to 1-butanol was studied over MgO-Al2O3 mixed oxide-based catalysts. Relationships between acid-base and catalytic properties and the effect of active metal on the hydrogen transfer reaction steps were investigated. The acid-base properties were studied by temperature-programmed desorption of CO2 and NH3 and by the FT-IR spectroscopic examination of adsorbed pyridine. Dispersion of the metal promoter (Pd, Pt, Ru, Ni) was determined by CO pulse chemisorption. The ethanol coupling reaction was studied using a flow-through microreactor system, He or H2 carrier gas, WHSV = 1 gEtOH·gcat.-1·h-1, at 21 bar, and 200-350 °C. Formation and transformation of surface species under catalytic conditions were studied by DRIFT spectroscopy. The highest butanol selectivity and yield was observed when the MgO-Al2O3 catalyst contained a relatively high amount of strong-base and medium-strong Lewis acid sites. The presence of metal improved the activity both in He and H2; however, the butanol selectivity significantly decreased at temperatures ≥ 300 °C due to acceleration of undesired side reactions. DRIFT spectroscopic results showed that the active metal promoted H-transfer from H2 over the narrow temperature range of 200-250 °C, where the equilibrium allowed significant concentrations of both dehydrogenated and hydrogenated products.

Enzymatic hydrolysate of water hyacinth with NaOH pretreatment for biobutanol production via ABE fermentation by Clostridium beijerinckii JCM 8026

Water hyacinth (WH; Eichhornia crassipes) is an invasive and free-floating aquatic plant that is rapidly spreading over the world causing various environmental problems. The utilization of WH as a feedstock for biofuel production is a challenging solution to overcome the environmental problem as well as address the energy shortage. Due to the complex structure of lignocellulosic materials, pretreatment is an inevitable step to maximize the sugar yield before being used as a substrate in fermentation. The aim of this study was to investigate the utilization of WH for biobutanol production via acetone-butanol-ethanol fermentation by Clostridium beijerinckii JCM 8026. The WH was pretreated with sodium hydroxide (NaOH) at various concentrations [0, 2, and 4% (w/v)], reaction times (30, 60, and 90 min), and temperatures (80, 100, and 120 °C). The pretreatment results were optimized using response surface methodology with a Box-Behnken design and Minitab software analysis. The maximum total sugar concentration (21.05 g/L) following cellulose digestion was obtained after a 2% (w/v) NaOH for 90 min at 100 °C. This hydrolysate slurry gave a subsequent butanol concentration, yield, and productivity of 2.09 g/L, 0.15 g/g, and 0.012 g/L/h, respectively, following C. beijerinckii JCM 8026 fermentation. To help understand the effect of the pretreatments, changes in the pretreated samples were observed by surface area analyses, scanning electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, and thermogravimetric analysis.

Highly selective butanol production by manipulating electron flow via cathodic electro-fermentation

Butanol production by solventogenic Clostridia shows great potential to combat the energy crisis, but is still challenged by low butanol selectivity and high downstream cost. In this study, a novel cathodic electro-fermentation (CEF) system mediated by methyl viologen (MV) was proposed and sequentially optimized to obtain highly selective butanol production. Under the optimal conditions (−0.60 V cathode potential, 0.50 mM MV, 30 g/L glucose), 7.17 ± 0.55 g/L butanol production were achieved with the yield of 0.32 ± 0.02 g/g. With the supplement of 4 g/L butyric acid as co-substrate, butanol production further improved to 13.14 ± 1.14 g/L with butanol yield and selectivity as high as 0.43 ± 0.01 g/g and 90.44 ± 1.66%, respectively. The polarized electrode enabled the unbalanced fermentation towards butanol formation and MV further inhibited hydrogen production, both of which contributed to the high-level butanol production and selectivity. The MV-mediated CEF system is a promising approach for cost-effective bio-butanol production.

Technical, Environmental, and Process Safety Assessment of Acetone-Butanol-Ethanol Fermentation of Cassava Residues

The North-Colombian region has enormous potential for producing bioproducts and bioenergy from agricultural residues. Yet, scaling bioproducts and bioenergy to industrial practice requires further investigation, especially for environmental impact minimization and improved process safety. This work assesses two alternatives for valorizing cassava residues via acetone, butanol, and ethanol (ABE) fermentation. Two ABE fermentation routes are assessed. In Route 1, pretreatment and purification involve dilute-acid pretreatment and multi-effect distillation and decantation operations, while Route 2 includes steam explosion and reactive distillation. Hazard Identification and Risk Assessment (HIRA) and Waste Reduction Algorithm (WAR) were applied to assess ABE fermentation. Simulation results indicate butanol yields of 0.10–0.12 kg/kg feedstock and net energy ratio (NER) <1. Route 2 shows the highest total output of Potential Environmental Impacts (PEI) with 5.56 PEI/kg butanol. Both ABE fermentation routes obtained Fire and Explosion Damage Index (FEDI) values above 300 for acetone and ethanol recovery/purification stages. Both routes are classified as “hazardous” considering the flammability of handled substances, and their relative safety performance is remarkably similar. These results pave the way toward deploying both routes for adding value to the cassava residues in North Colombia by applying safe, efficient, and environmentally friendly transformation technologies.

Construction and metabolic analysis of acetone-butanol-ethanol fermentation using mixed acetic acid and lactic acid in wastewater

To reuse organic acids in wastewater, efficient acetone-butanol-ethanol (ABE) fermentation was constructed using mixed acetic acid and lactic acid. The mixed acids were co-consumed with glucose, producing higher butanol concentration (6.96 g/L) and butanol yield (0.494 C-mol/C-mol) than using each single acid (6.22 g/L and 0.432 C-mol/C-mol). This result was verified by 13 C tracer experiment that addition of lactic acid improved carbon distribution to butanol instead of acetone , while acetic acid was readily consumed than lactic acid at initial fermentation stage. Subsequently, pH-stat fed batch fermentation at low pH of 5.5 further improved acid consumptions (6.34 g/L acetic acid and 9.06 g/L lactic acid) and solvent productions (12.4 g/L butanol and 6.50 g/L acetone). Finally, ABE fermentation using wastewater of lactic acid fermentation was performed without inhibition. Thus, efficient ABE fermentation can be implemented using mixed acetic acid and lactic acid, which is superior to using single acid. • This is the first study using mixed acetate and lactate for ABE fermentation. • 13 C experiment verified the metabolic flux to butanol was improved by the acids. • pH and glucose played an important role in acid consumption and solvent production. • Fermentation from lactic acid fermentation broth was performed without inhibition.

Techno-Economic Analysis of Extractive Butanol Fermentation by Immobilized Cells with Large Extractant Volume

There are several challenges for ABE fermentation to be used in an industrial scale including the low of butanol yield, the high energy requirement for separation and purification, and the competeness of sugar with food demand as substrat. In this study, techno-economical aspects of ABE fermentation by using immobilized cells with large extractant volume were studied. Overall production process was designed using rice straw as raw material which is semi-hydrolyzed to produce cellobiose, glucose, xylose, and arabinose mixture. Concentrated sugar was then fed to extractive fed-batch fermentation using immobilized cells. Finally, extractant was recovered and products were purified by distillation column. By evaluating this process design for the small scale capacity of 238 kg-butanol and acetone/day, the energy requirement was 41.3 MJ/kg-butanol and acetone and the cost was 1.91 $/kg-butanol and acetone. Although the cost was higher than butanol produced by petrochemical process of 1.08 $/kg-butanol, it may reduce if the scale is increased.

Butanol production from Thai traditional beverage (Sato) factory wastewater using newly isolated Clostridium beijerinckii CUEA02

Sato, a Thai traditional beverage, wastewater (STW) contains residual rice constituents with high chemical and biological oxygen demands, and so its disposal is an environmental problem. To solve this, the potential of STW for butanol production, which has never been evaluated for use in ABE fermentation, is reported. Clostridium beijerinckii CUEA02, the strain producing the highest butanol level in this study, was isolated from natural sources in Thailand. Isolate CUEA02 was closely related to C. beijerinckii with an average nucleotide identity of 95.14% and could grow and produce butanol from various carbon sources. The butanol production was 8.32 ± 0.08 g/L in flask fermentation under an initial pH of 6.5, 35 °C, 10% (v/v) inoculum size, and 50 g/L of glucose. To evaluate the potential of STW, it was diluted 0.1x, 0.2x, 0.3x, and 0.4x for ABE production by CUEA02, with the 0.1x dilution giving the highest butanol yield (0.23 g/g). To improve the butanol production, the initial pH was adjusted from 4.75 to 6.5, resulting in a 2.13-fold enhanced butanol yield (0.49 g/g). Moreover, the addition of yeast extract at 2 g/L increased the butanol production by up to 29% in STW using C. beijerinckii CUEA02.

Formation and multiple catalysis of copper-cysteine complex enhance butanol fermentation from rice straw

Butanol production from rice straw via Acetone-Butanol-Ethanol (ABE) fermentation has been considered a sustainable and cost-effective approach to cope with energy crisis and environmental issues. However, a number of factors, particularly low butanol production and energy-intensive separation, impede its industrial application. In this study, Cu2+, Zn2+, Fe2+, and Ni2+ were individually induced into the ABE fermentation system of Clostridium beijerinckii NCIMB 8052 with butyric acid and sugars both from rice straw as co-substrates to increase the specific butanol yield. The results demonstrated that the additional 0.20 g/L Cu2+ (i.e. 1.25 mmol/L) significantly increased SBY and butanol/acetone to 0.194 ± 0.006 g/g rice straw and 3.90 ± 0.21, respectively, compared to those of 0.126 ± 0.004 g/g rice straw and 2.61 ± 0.17 in control system. The improved butanol production was attributed to the formation of Cu2+-cysteine/Cu+-cysteine complex, which enhanced the adsorption of butyrate from rice straw by upgrading the activity of key enzyme (ctfAB) and NADH/NAD+. The study provides a reinforcement strategy for efficient lignocellulosic butanol production.

Enhancement of butanol production in Clostridium acetobutylicum SE25 through oxidation-reduction potential regulation and analysis of its metabolic mechanisms

In this study, an exogenous regulatory strategy named as oxidation-reduction potential (ORP) regulation was performed in the butanol biosynthesis process by Clostridium acetobutylicum SE25 to accelerate the carbon metabolism flow to butanol and enhance butanol production. The findings show that the dithiothreitol and potassium ferricyanide were the optimal redox agents for regulating the ORP of the fermentation system. Specifically, when the ORP value of fermentation broth was controlled at −400 mV, the highest production of butanol (14.30 ± 0.65 g/L) was obtained, which was 10.80 % higher than that of the control group. And the yield and productivity of butanol were 0.24 g/g and 0.24 g/L/h, respectively. Furthermore, the physiological mechanisms of the effects of different ORP levels on butanol synthesis was investigated, and the results demonstrate that ORP could both regulate the level and ratio of energy charge and NADH/NAD+, and redistribute metabolic flux. Thus, the ORP control strategy, according to the results in this study, is an effective and attainable approach to obtain the high production of butanol.

Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum

Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP+ reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD+ reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain.

Technoeconomic analysis and environmental sustainability estimation of bioalcohol production from barley straw

European Union bioeconomy policy emphasizes that the production of renewable transportation fuels should replace fossil fuels as much as possible. In particular, the utilization of waste or side-stream lignocellulosic materials for fuel production is highly recommended. Moreover, future crises forcing a reliance on locally available sources for fuels and energy may become increasingly common. Barley straw, a common agricultural residue in northern Europe, is a potential raw material for bioalcohol production via fermentation. In this study, the technoeconomic and environmental sustainability of bioethanol and biobutanol production from barley straw were evaluated. When compared with fossil gasoline production and use, the greenhouse gas emissions reduction 77.6% and 72.1% were achieved for ethanol and butanol production, respectively. Thus, the emission reduction of 65% for biofuels demanded by the European Union renewable energy directive was achieved in both biofuel production processes evaluated. However, our results indicated that ethanol production from barley straw, a well-known and mature technology, was an economically feasible process (NPV positive, IRR 20%) but that butanol production with Clostridium species through acetone-butanol-ethanol fermentation has still technoeconomic challenges to overcome (NPV negative, IRR below 10%). This was mainly due to the low yield and high recovery costs of butanol.

Effectiveness of Low-Cost Bioreactors Integrated with a Gas Stripping System for Butanol Fermentation from Sugarcane Molasses by Clostridium beijerinckii

The effectiveness of column bioreactors for butanol fermentation from sugarcane molasses by Clostridium beijerinckii TISTR 1461 was investigated. This fermentation was operated at an initial pH of 6.5 and temperature of 37 °C under anaerobic conditions. A 1-L bubble column bioreactor was used with various gas circulation rates ranging from 0.2 to 1.0 L/min. The highest butanol concentration (PB, 8.72 g/L), productivity (QB, 0.24 g/L∙h) and yield (YB/S, 0.21 g/g) were obtained with a gas circulation of 0.2 L/min. To improve butanol production efficiency, gas-lift column bioreactors with internal and external loops at 0.2 L/min of circulating gas were used. Higher PB (10.50–10.58 g/L), QB (0.29 g/L∙h) and YB/S (0.22–0.23 g/g) values were obtained in gas-lift column bioreactors. These values were similar to those using a more complex 2-L stirred-tank bioreactor (PB, 10.10 g/L; QB, 0.28 g/L h and YB/S, 0.22 g/g). Hence, gas-lift column bioreactors have potential for use as low-cost fermenters instead of stirred-tank bioreactors for butanol fermentation. When the gas-lift column bioreactor with an internal loop was coupled with a gas stripping system, it yielded an enhanced PB and sugar consumption of approximately 9% and 7%, respectively, compared to a system with no gas stripping.

Butanol Production by a Novel Efficient Method Using Mixed Cultures of Clostridium beijerinckii and Arthrobacter sp. in Stirred-Tank and Gas-Lift Bioreactors

Arthrobacter sp. BCC 72131, an obligate aerobic bacterium, was used to create anaerobic conditions, and Clostridium beijerinckii TISTR 1461 was used as a butanol producer in an acetone-butanol–ethanol (ABE) fermentation. Sweet sorghum juice (SSJ) medium containing 60 g/L of total sugar supplemented with 1.27 g/L of (NH4)2SO4 was used as a butanol production (BP) medium. Arthrobacter sp. was inoculated into the BP medium in 1-L screw-capped bottles. After 2, 4, 6 and 12 h of Arthrobacter sp. cultivation at 30 °C, C. beijerinckii was transferred into the BP medium to start butanol production at 37 °C. The results showed that C. beijerinckii inoculation after 6 h of Arthrobacter sp. cultivation gave the highest butanol titer (PB) at 12.56 g/L, with a butanol yield (YB/S) and volumetric butanol productivity (QB) of 0.34 g/g and 0.23 g/L·h, respectively. These values are approximately 10–27% higher than those of the control experiment using a single culture of C. beijerinckii TISTR 1461 and oxygen-free nitrogen (OFN) gas flushing to create anaerobic conditions. Field emission scanning electron microscopic (FE-SEM) images of Clostridium cells, as well as protein and free amino nitrogen concentrations in the broth during butanol fermentation were also studied to confirm the results. The butanol fermentation was then carried out in a 5.6-L stirred-tank and a 1.2-L low-cost gas-lift bioreactor by the mixed cultures using the optimal time of Clostridium inoculation. The PB, YB/S and QB values obtained were not significantly different from those in the 1-L screw-capped bottles. Hence, Arthrobacter sp. can be used as a novel method to create anaerobic conditions instead of a traditional method employing OFN gas flushing. Using mixed cultures of Arthrobacter sp. BCC 72131 and C. beijerinckii TISTR 1461 is a practical method to produce butanol on a large-scale, both in complex and low-cost bioreactors.

Engineering E. coli to synthesize butanol.

Biobutanol is gaining much attention as a potential biofuel due to its superior properties over ethanol. Butanol has been naturally produced via acetone-butanol-ethanol (ABE) fermentation by many Clostridium species, which are not very user-friendly bacteria. Therefore, to improve butanol titers and yield, various butanol synthesis pathways have been engineered in Escherichia coli, a much more robust and convenient host than Clostridium species. This review mainly focuses on the biosynthesis of n-butanol in engineered E. coli with an emphasis on efficient enzymes for butanol production in E. coli, butanol competing pathways, and genome engineering of E. coli for butanol production. In addition, the use of alternate strategies for butanol biosynthesis/enhancement, alternate substrates for the low cost of butanol production, and genetic improvement for butanol tolerance in E. coli have also been discussed.