N-caproic Acid Research Articles

Biochar confers significant microbial resistance to ammonia toxicity in n-caproic acid production

Microbial chain elongation integrating innovative bioconversion technologies with organic waste utilization can transition current energy-intensive n-caproic acid production to sustainable circular bioeconomy systems. However, ammonia-rich waste streams, despite their suitability, pose inhibitory challenges to these bioconversion processes. Herein, biochar was employed as an additive to enhance the activity of chain elongating microbes under ammonia inhibition conditions, with an objective to detail underlying mechanisms of improvements. Biochar addition significantly improved chain elongation performance even under severe ammonia stress (exceeding 8 g N/L), increasing n-caproic acid yields by 40 % to 158 % and reducing lag times by 51 % to 90 %, compared with the best-performing group without biochar addition. The material contribution to n-caproic production reached up to 94.3 % (at 4 g N/L). These enhancements were mainly attributed to the new electron syntrophy induced by biochar, which improved electron transfer system activity and electrical conductivity of the fermentation system. This is further substantiated by increased relative abundances of the genus Sporanaerobacter, electroactive bacteria, and up-regulated direct electron transfer-related genes including conductive pili and c-type cytochrome. This study demonstrates that biochar can confer robust resilience to ammonia toxicity in functional microbes, paving a way for efficient and sustainable n-caproic acid production.

Nano zero valent iron stimulated acetaldehyde oxidation, electron transfer, and RBO pathway for enhanced medium-chain carboxylic acids production

Nano zero-valent iron (NZVI) has been shown to effectively enhance the chain elongation (CE) process, addressing the issue of limited yield of medium-chain carboxylic acids (MCCA) from organic wastewater. However, the specific impact of NZVI on the metabolism of CE bacteria (CEB) is not well understood. In this study, it was aimed to investigate the mechanism by which an optimal concentration of NZVI influences CE metabolism, particularly in relation to ethanol oxidation, electron transfer, and MCCA synthesis. This was achieved through single-factor influence experiments and metagenomic analysis. The results showed that the addition of 1 g/gVSS NZVI achieved the highest MCCA yield (n-caproic acid + n-octanoic acid) at 2.02 g COD/L, which was 4.9 times higher than the control. This improvement in MCCA production induced by NZVI was attributed to several factors. Firstly, NZVI facilitated the oxidation of acetaldehyde, leading to its reduced accumulation in the system (from 18.4 % to 5.8 %), due to the optimized chemical environment created by NZVI corrosion, including near-neutral pH and a more reductive oxidation–reduction potential (ORP). Additionally, the inherent conductivity property of NZVI and the additional Fe ions released during corrosion improved the electron transfer efficiency between CEB. Lastly, both the composition of microbial communities and the abundance of unique enzyme genes confirmed the selective stimulation of NZVI on the reverse β-oxidation (RBO) pathway. These findings provide valuable insights into the role of NZVI in CEB metabolism and its potential application for enhancing MCCA production in CE bioreactors.

Analysis of the performance and microbial community of continuous dark fermentation combined with steady static magnetic field

The effect of external static magnetic field on hydrogen production and its mechanism were studied by continuous dark fermentation with no pretreatment excess sludge as inoculum. The results showed that under conditions of 37 ± 1 ℃, HRT 8 h, OLR 15 g/(L· d) and no control of influent pH, 78 mT external static magnetic field by adding magnets on both sides of the reactor could accelerate the enrichment of Ethanoligenens and improve the activity of reduced coenzyme I (96 %), ferredoxin oxidoreductase (29 %) and pyruvate: ferredoxin oxidoreductase (7 %) so that the maximum hydrogen productivity could reach 1.657 L H2/L, which was 5 times higher than that of non-magnetic group. Moreover, the n-caproic acid production could also increase due to the enrichment of carbon chain elongation microorganisms. The decline of hydrogen production was mainly due to hydrogenotrophic methanogenesis and acetic acid accumulation. This study was benefit to understand the interaction mechanism between magnetic field and microorganisms during the process of biological hydrogen production.

Life cycle assessment of n-caproic acid production via chain elongation from food waste: Comparison of shunting and staged technology

n-Caproic acid is a widely used biochemical that can be produced from organic waste through chain elongation technology. This study aims to evaluate the environmental impacts of n-caproic acid production through chain elongation by two processes (i.e., shunting and staged technology). The Open-life cycle assessment (LCA) model was used to calculate the environmental impacts of both technologies based on experimental data. Results showed that the shunting technology had higher environmental impacts than the staged technology. Water and electricity made bigger contribution to the environmental impacts of both technologies. Reusing chain elongation effluent substituting for water and using electricity produced by wind power could reduce the environmental impacts of water and electricity effectively. Using ethanol from food waste had higher global warming potential than fossil ethanol, which suggested that a cradle-to-grave LCA is needed to be carried out for specific raw materials and chain elongation products in the future.

Physicochemical Features and Volatile Organic Compounds of Horse Loin Subjected to Sous-Vide Cooking.

The purpose of this study was to evaluate the effect of temperature and time of sous-vide cooking method on the characteristics of Thoroughbred horse loin. Sliced portions (200 ± 50 g) were cooked by boiling (control) and sous-vide (65 and 70 °C for 12, 18, and 24 h). The samples were analyzed for proximate composition, pH, color, texture, microstructure, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), microbiology, volatile organic compounds (VOCs), nucleotide content, and fatty acids composition. The color analysis showed decreased redness at elevated temperatures. Improved tenderness, demonstrated by reduced shear force values (36.36 N at 65 °C for 24 h and 35.70 N at 70 °C for 24 h). The micrographs indicated dense fiber arrangements at 70 °C. The SDS-PAGE revealed muscle protein degradation with extended sous-vide cooking. The VOC analysis identified specific compounds, potentially distinctive markers for sous-vide cooking of horse meat including 1-octen-3-ol, decanal, n-caproic acid vinyl ester, cyclotetrasiloxane, octamethyl, and 3,3-dimethyl-1,2-epoxybutane. This study highlights the cooking time's primary role in sous vide-cooked horse meat tenderness and proposes specific VOCs as potential markers. Further research should explore the exclusivity of these VOCs to sous-vide cooking.

Biogas slurry recirculation regulates food waste fermentation: Effects and mechanisms

Regularly adding biogas slurry into fermentation reactors is an effective way to enhance hydrogen or methane production. However, how this method affects the production of valuable organic acids and alcohols is still being determined. This study investigated the effects of different addition ratios on semi-continuous fermentation reactors using food waste as a substrate. The results showed that an addition ratio of 0.2 increased lactic acid production by 30% with a yield of 0.38 ± 0.01 g/g VS, while a ratio of 0.4 resulted in mixed acid fermentation dominated by n-butyric acid (0.07 ± 0.01 g/g VS) and n-caproic acid (0.06 ± 0.00 g/g VS). The introduction of Bifidobacteriaceae by biogas slurry played a crucial role in increasing lactic acid production. In contrast, exclusive medium-chain fatty acid producers enhanced the synthesis of caproic acid and heptanoic acid via the reverse β-oxidation pathway. Mechanism analyses suggested that microbial community structure and activity, substrate hydrolysis, and cell membrane transport system and structure changed to varying degrees after adding biogas slurry.

A preliminary study on the feasibility of industrialization for n-caproic acid recovery from food wastewater: From lab to pilot

Food wastewater is associated with greenhouse gas emission and has a significant water footprint. Here, the platform chemical n-caproate was recovered from liquor brewing wastewater at maximum and mean concentrations of 26.4 g/L and 17.0 ± 4.3 g/L, respectively, after 377 d operation. Laboratory-scale lactate-driven chain elongation (CE) process was implemented first. Taxonomic composition and metagenomic data analyses revealed that Caproiciproducens (e.g., Ruminococcaceae bacterium CPB6) and bacteria affiliated with Lachnospiraceae transformed lactate to n-caproate by reverse β-oxidation and/or fatty acid biosynthesis. The lactate-driven CE process was then scaled up from 2.5 L to 500 L and achieved a n-caproate production of 14.5 ± 0.6 g/L within 96 h. n-Caproic acid was extracted at a concentration and purity of 815.9 ± 8.3 g/L and 88.6 ± 8.9 %, respectively. The present study demonstrated a commercially viable strategy for resource recovery and carbon fixation from food waste streams.

Promoting chain elongation efficiency from food waste by refluxing chain elongation fermentation liquid

Food waste fermentation can produce medium chain fatty acids (MCFAs) through chain elongation, but n-butyric acid normally accounts for the largest proportion of products. In order to fully utilize these n-butyric acid to produce MCFAs, reflux was carried out and five reflux ratios were studied. Results showed that reflux effectively alleviated the inhibitory effect of undissociated n-butyric acid and enhanced the production of MCFAs, the optimum reflux ratio was 100%, n-caproic acid production increased by 707.43 ± 39.54 mg COD/g VS, which was 4.17 times higher than the control without reflux. Reflux can also promote the production of n-valeric acid and heptylic acid. The kinetic analysis also confirmed that 100% was the optimum reflux ratio for improving MCFA production, and the carbon proportion and electron transfer efficiency of n-caproic acid were 45.40% and 85.50% respectively. By refluxing, the microbiomes related to the MCFA production were further enriched. Mass balance analysis of n-caproic acid showed that 100% reflux ratio resulted in a production of n-caproic acid from 88.57 to 152.57 g/kg dry food waste. This study offers a new direction for efficiently utilizing the food waste and a new perspective for improving the production of MCFAs.

Caproic Acid-Producing Bacteria in Chinese Baijiu Brewing

Caproic acid can be used as spices, preservatives, animal feed additives, and biofuels. At the same time, caproic acid plays an important role in Chinese Baijiu. It is the precursor substance for the synthesis of ethyl caproate, which directly affects the quality of Chinese Baijiu. Caproic acid-producing bacteria are the main microorganisms that synthesize caproic acid in Chinese Baijiu, and the most common strain is Clostridium kluyveri. Caproic acid-producing bacteria synthesize n-caproic acid through reverse β-oxidation to extend the carboxylic acid chain. This method mainly uses ethanol and lactic acid as substrates. Ethanol and lactic acid are converted into acetyl-CoA, and acetyl-CoA undergoes a series of condensation, dehydrogenation, dehydration, and reduction to extend the carboxylic acid chain. This review addresses the important issues of caproic acid-producing bacteria in the brewing process of Baijiu: the common caproic acid-producing bacteria that have been reported metabolic pathways, factors affecting acid production, biological competition pathways, and the effect of mixed bacteria fermentation on acid production. It is hoped that this will provide new ideas for the study of caproic acid-producing bacteria in Chinese Baijiu.

Thermodynamic approach to foresee experimental CO2 reduction to organic compounds

Anaerobic gas fermentation is a promising approach to transform carbon dioxide (CO2) into chemical building blocks. However, the main operational conditions to enhance the process and its selectivity are still unknown. The main objective of this study was to trigger chain elongation from a joint perspective of thermodynamic and experimental assessment. Thermodynamics revealed that acetic acid formation was the most spontaneous reaction, followed by n-caproic and n-butyric acids, while the doorway for alcohols production was bounded by the selected conditions. Best parameters combinations were applied in three 0.12 L fermenters. Experimentally, n-caproic acid formation was boosted at pH 7, 37 °C, Acetate:Ethanol mass ratio of 1:3 and low H2 partial pressure. Though these conditions did not match with those required to produce their main substrates, the unification of both perspectives yielded the highest n-caproic acid concentration (>11 g L−1) so far from simple substrates, accounting for 77 % of the total products.

Effect of Static Magnetic Field on Hydrogen and N-Caproic Acid Production by Dark Fermentation with No Pretreatment Excess Sludge as Inoculum

Effect of Static Magnetic Field on Hydrogen and N-Caproic Acid Production by Dark Fermentation with No Pretreatment Excess Sludge as Inoculum

Reactor microbiome enriches vegetable oil with n-caproate and n-caprylate for potential functionalized feed additive production via extractive lactate-based chain elongation

BackgroundBiotechnological processes for efficient resource recovery from residual materials rely on complex conversions carried out by reactor microbiomes. Chain elongation microbiomes produce valuable medium-chain carboxylates (MCC) that can be used as biobased starting materials in the chemical, agriculture and food industry. In this study, sunflower oil is used as an application-compatible solvent to accumulate microbially produced MCC during extractive lactate-based chain elongation. The MCC-enriched solvent is harvested as a potential novel product for direct application without further MCC purification, e.g., direct use for animal nutrition. Sunflower oil biocompatibility, in situ extraction performance and effects on chain elongation were evaluated in batch and continuous experiments. Microbial community composition and dynamics of continuous experiments were analyzed based on 16S rRNA gene sequencing data. Potential applications of MCC-enriched solvents along with future research directions are discussed.ResultsSunflower oil showed high MCC extraction specificity and similar biocompatibility to oleyl alcohol in batch extractive fermentation of lactate and food waste. Continuous chain elongation microbiomes produced the MCC n-caproate (nC6) and n-caprylate (nC8) from l-lactate and acetate at pH 5.0 standing high undissociated n-caproic acid concentrations (3 g L−1). Extractive chain elongation with sunflower oil relieved apparent toxicity of MCC and production rates and selectivities reached maximum values of 5.16 ± 0.41 g nC6 L−1 d−1 (MCC: 11.5 g COD L−1 d−1) and 84 ± 5% (e− eq MCC per e− eq products), respectively. MCC were selectively enriched in sunflower oil to concentrations up to 72 g nC6 L−1 and 3 g nC8 L−1, equivalent to 8.3 wt% in MCC-enriched sunflower oil. Fermentation at pH 7.0 produced propionate and n-butyrate instead of MCC. Sunflower oil showed stable linoleic and oleic acids composition during extractive chain elongation regardless of pH conditions. Reactor microbiomes showed reduced diversity at pH 5.0 with MCC production linked to Caproiciproducens co-occurring with Clostridiumtyrobutyricum, Clostridiumluticellarii and Lactobacillus species. Abundant taxa at pH 7.0 were Anaerotignum, Lachnospiraceae and Sporoanaerobacter.ConclusionsSunflower oil is a suitable biobased solvent to selectively concentrate MCC. Extractive reactor microbiomes produced MCC with improved selectivity and production rate, while downstream processing complexity was reduced. Potential applications of MCC-enriched solvents may include feed, food and biofuels purposes.

Genome-Wide Transcriptomic Analysis of n-Caproic Acid Production in Ruminococcaceae Bacterium CPB6 with Lactate Supplementation.

n-Caproic acid (CA) is gaining increased attention due to its high value as a chemical feedstock. Ruminococcaceae bacterium strain CPB6 is an anaerobic mesophilic bacterium that is highly prolific in its ability to perform chain elongation of lactate to CA. However, little is known about the genome-wide transcriptional analysis of strain CPB6 for CA production triggered by the supplementation of exogenous lactate. In this study, cultivation of strain CPB6 was carried out in the absence and presence of lactate. Transcriptional profiles were analyzed using RNA-seq, and differentially expressed genes (DEGs) between the lactate-supplemented cells and control cells without lactate were analyzed. The results showed that lactate supplementation led to earlier CA p,roduction, and higher final CA titer and productivity. 295 genes were substrate and/or growth dependent, and these genes cover crucial functional categories. Specifically, 5 genes responsible for the reverse β-oxidation pathway, 11 genes encoding ATP-binding cassette (ABC) transporters, 6 genes encoding substrate-binding protein (SBP), and 4 genes encoding phosphotransferase system (PTS) transporters were strikingly upregulated in response to the addition of lactate. These genes would be candidates for future studies aiming at understanding the regulatory mechanism of lactate conversion into CA, as well as for the improvement of CA production in strain CPB6. The findings presented herein reveal unique insights into the biomolecular effect of lactate on CA production at the transcriptional level.

Selecting fermentation products for food waste valorisation with HRT and OLR as the key operational parameters

Acidogenic fermentation is attractive for food waste valorisation. A better understanding is required on how operation affects product selectivity. This study demonstrated that the hydraulic retention time (HRT) and organic loading rate (OLR) selected fermentation pathways in a single-stage, semi-continuous stirred tank reactor. Three combinations of HRT and OLR were tested to distinguish the effect of each parameter. Three fermentation profiles with distinct microbial communities were obtained. Predominantly n-butyric acid (13 ± 2 gCOD L−1, 55 ± 14% of carboxylates) was produced at an HRT of 8.5 days and OLR around 12 gCOD L−1d−1. Operating at an HRT two days longer, yet with similar OLR, stimulated chain elongation (up to 13.6 gCOD L−1 of n-caproic acid). This was reflected by a microbial community twice as diverse at longer HRT as indicated by first and second order Hill number (1D = 24 ± 4, 2D = 12 ± 3) and by a higher relative abundance of genera related to secondary fermentation, such as the VFA-elongating Caproiciproducens spp., and secondary lactic acid fermenter Secundilactobacillus spp.. Operating at a higher OLR (20 gCOD L−1d−1) but HRT of 8.5 days, resulted in typical lactic acid fermentation (34 ± 5 gCOD L−1) harbouring a less diverse community (1D = 8.0 ± 0.7, 2D = 5.7 ± 0.9) rich in acid-resistant homofermentative Lactobacillus spp. These findings demonstrate that a flexible product portfolio can be achieved by small adjustments in two key operating conditions. This improves the economic potential of acidogenic fermentation for food waste valorisation.

The Isolate Caproiciproducens sp. 7D4C2 Produces n-Caproate at Mildly Acidic Conditions From Hexoses: Genome and rBOX Comparison With Related Strains and Chain-Elongating Bacteria.

Bulk production of medium-chain carboxylates (MCCs) with 6–12 carbon atoms is of great interest to biotechnology. Open cultures (e.g., reactor microbiomes) have been utilized to generate MCCs in bioreactors. When in-line MCC extraction and prevention of product inhibition is required, the bioreactors have been operated at mildly acidic pH (5.0–5.5). However, model chain-elongating bacteria grow optimally at neutral pH values. Here, we isolated a chain-elongating bacterium (strain 7D4C2) that grows at mildly acidic pH. We studied its metabolism and compared its whole genome and the reverse β-oxidation (rBOX) genes to other bacteria. Strain 7D4C2 produces lactate, acetate, n-butyrate, n-caproate, biomass, and H2/CO2 from hexoses. With only fructose as substrate (pH 5.5), the maximum n-caproate specificity (i.e., products per other carboxylates produced) was 60.9 ± 1.5%. However, this was considerably higher at 83.1 ± 0.44% when both fructose and n-butyrate (electron acceptor) were combined as a substrate. A comparison of 7D4C2 cultures with fructose and n-butyrate with an increasing pH value from 4.5 to 9.0 showed a decreasing n-caproate specificity from ∼92% at mildly acidic pH (pH 4.5-5.0) to ∼24% at alkaline pH (pH 9.0). Moreover, when carboxylates were extracted from the broth (undissociated n-caproic acid was ∼0.3 mM), the n-caproate selectivity (i.e., product per substrate fed) was 42.6 ± 19.0% higher compared to 7D4C2 cultures without extraction. Based on the 16S rRNA gene sequence, strain 7D4C2 is most closely related to the isolates Caproicibacter fermentans (99.5%) and Caproiciproducens galactitolivorans (94.7%), which are chain-elongating bacteria that are also capable of lactate production. Whole-genome analyses indicate that strain 7D4C2, C. fermentans, and C. galactitolivorans belong to the same genus of Caproiciproducens. Their rBOX genes are conserved and located next to each other, forming a gene cluster, which is different than for other chain-elongating bacteria such as Megasphaera spp. In conclusion, Caproiciproducens spp., comprising strain 7D4C2, C. fermentans, C. galactitolivorans, and several unclassified strains, are chain-elongating bacteria that encode a highly conserved rBOX gene cluster. Caproiciproducens sp. 7D4C2 (DSM 110548) was studied here to understand n-caproate production better at mildly acidic pH within microbiomes and has the additional potential as a pure-culture production strain to convert sugars into n-caproate.

DNA barcoding and FAME profiling divulges the soil marine Bacillus licheniformis as a potential candidate for n-caproic acids production

DNA barcoding and FAME profiling divulges the soil marine Bacillus licheniformis as a potential candidate for n-caproic acids production

Production and extraction of medium chain carboxylic acids at a semi-pilot scale

Abstract Microbial chain elongation enables the conversion of organic wastes and agroindustrial sidestreams into medium chain carboxylic acids (MCCAs), with application in the production of plasticizers, personal care products, functional feed additives, and fuels. This work describes the up-scaling of the production of MCCAs from thin stillage, and their separation into an oil at kilogram scale with a MCCA content of more than 72%. The chain elongation occurred at a maximum rate of 1.7 ± 0.6 g MCCA L−1 d−1 without the use of ethanol as electron donor, even when it was supplied. Two in-line extraction trains are compared: (1) solvent extraction based on pertraction followed by electrochemical phase separation of the acids through a 2-compartment membrane electrolysis (ME) cell, and (2) direct electrochemical extraction from the fermentation broth using a 3-compartment ME cell. Using the first treatment train, n-caproic acid was extracted at an efficiency of 73%. The decrease in the fermentation pH from 5.5 to 5.0 enabled doubling the MCCA and oil production rate without a concomitant increase in NaOH dosage for pH stabilization. The selectivity brought about by pertraction and its capacity to up-concentrate MCCAs in an alkaline-pH extract enabled a more efficient use of the electricity for ME (as low as 6.2 kWh kg−1 oil) when compared to a period when ME was applied directly to the filtered fermentation broth (160 kWh kg−1 oil). The 3-compartment ME cell was able to phase separate oil extracting directly from the fermentation broth but it was not selective for longer-chain carboxylic acids due to co-extraction of short chain carboxylic acids and inorganic anions.

Cloning and characterization of a L-lactate dehydrogenase gene from Ruminococcaceae bacterium CPB6.

Lactate are proved to be attractive electron donor for the production of n-caproic acid (CA) that is a high value-added fuel precursor and chemical feedstock, but little is known about molecular mechanism of lactate transformation. In the present study, the gene for L-lactate dehydrogenase (LDH, EC.1.1.1.27) from a Ruminococcaceae strain CPB6 was cloned and expressed in Escherichia coli BL21 (DE3) with plasmid pET28a. The recombinant LDH exhibited molecular weight of 36-38kDa in SDS-PAGE. The purified LDH was found to have the maximal oxidation activity of 29.6 U/mg from lactate to pyruvate at pH 6.5, and the maximal reduction activity of 10.4 U/mg from pyruvate to lactate at pH 8.5, respectively. Strikingly, its oxidative activity predominates over reductive activity, leading to a 17-fold increase for the utilization of lactate in E. coli/pET28a-LDH than E. coli/pET28a. The CPB6 LDH gene encodes a 315 amino acid protein sharing 42.19% similarity with Clostridium beijerinckii LDH, and lower similarity with LDHs of other organisms. Significant difference were observed between the CPB6 LDH and C. beijerinckii and C. acetobutylicum LDH in the predicted tertiary structure and active center. Further, X-ray crystal structure analysis need to be performed to verify the specific active center of the CPB6 LDH and its role in the conversion of lactate into CA.

Chain elongation performances with anaerobic fermentation liquid from sewage sludge with high total solid as electron acceptor

This work studied the effect of total solid (TS) of sewage sludge on VFA production and composition in anaerobic fermentation. Results revealed that VFA concentration reached the highest of 10.16 g/L and the ratio of acetic acid, propionic acid and n-butyric acid was 5:2:2 with the 8% TS sewage sludge. In subsequent chain elongation with sludge fermentation liquid, n-caproic acid concentration reached 43.45 mmol/L. The microbial community analysis indicated that relative abundance of Clostridium_sensu_stricto_12 for n-caproic acid production was high (52.41%). The chain elongation with sludge fermentation liquid had more pathways to produce n-caproic acid, and the chain elongation reactions were thermodynamically possible. The mixed VFAs and high concentration of n-butyric acid benefitted n-caproic acid production. Carbon balance revealed that the VFA composition of sludge fermentation liquid was beneficial to the chain elongation. This study will contribute to wasted sludge minimization and high-value material production.

Volumetric studies of solvation of carboxylic acids in aqueous, carbon tetrachloride, methanolic and methanolic-urea binary solutions at 298.15 K

Abstract Experimental measurements of density property at 298.15 K for aqueous, carbon tetrachloride, methanolic, and 3.0004 mol·kg−1 urea in methanol as solvent systems for binary solutions of carboxylic acids (propionic acid, n-butyric acid, n-valeric acid and n-caproic acid) are reported. The data of density are used to obtain apparent molar volume of acids at the finite concentrations of solute (Vϕ) which were appropriately extrapolated to infinitely dilute concentration of acid to obtain the limiting apparent molar volumes (Vϕ0). The calculations of volume changes due to dimerization equilibria (in carbon tetrachloride and aqueous solutions) are made and used to study the volume changes due to dimerization of acid molecules. The results of Vϕ in methanol-urea solvent system are explained on the basis of channel-like structure-formation for the species (urea and acid molecules) in solution phase. The negative volume changes observed in case of aqueous solutions are explained on the basis of hydrophobic hydration while the same in carbon tetrachloride solutions occurring with positive volume changes are interpreted in terms of hydrogen-bonded dimer formation equilibria. The Vϕ variation with acid concentrations has been explained on the basis of solute-solvent interactions foe studied acids in methanol while in aqueous solutions, the same has been attributed to ionization (limiting concentration range), hydrogen bonding and hydrophobic interactions.