Long-chain Fatty Acids Inhibition Research Articles

Methanogenic degradation of long-chain fatty acids associated with syntrophic microbial dynamics during thermophilic AnMBR treatment of lipid-rich dairy wastes

Anaerobic membrane reactors (AnMBRs) have been applied to effectively treat dairy wastes because they can address the drawbacks of sludge flotation and biomass washout. However, when dairy wastes contain considerable proportion of lipids, any anaerobic systems will likely encounter challenges with long-chain fatty acid (LCFA) inhibition. In this study, a thermophilic AnMBR was operated to treat dairy processing wastewater (DPW) plus ice cream waste (ICW). To evaluate the impact of LCFAs, the lipid-rich ICW was gradually introduced at different levels (0, 5, 10, and 20 %) into the AnMBR, along with a stepwise increase in lipid loading rate (LLR) from 0.22 to 11.24 g lipid/L/d. The results of biomethanation revealed that the AnMBR could be successfully operated when the LLR was maintained at 5.73 g lipid/L/d with a lipid/VS ratio of 50.22 % in the feedstock. However, at ultra-high LLR of 11.24 g lipid/L/d (20 % ICW added), a significant inhibitory effect on methane yields was observed, resulting in a reduction of 56.90 %. Dynamic analysis of the LCFAs profiled that a lower degradation rate was an important factor contributing to LCFA accumulation, and the inhibition concentration of various LCFAs was identified. Based on microbial evolution and co-occurrence network results, potential syntrophic partnerships between LCFA-degrading bacteria and methanogens were proposed. The responsible enzyme genes in the LCFA-degrading, acetoclastic, and hydrogenotrophic methanogenic pathways played crucial roles in LCFA inhibition and degradation. These findings offer practical insights for the efficient methanogenic treatment of lipid-rich wastes.

Oil in food waste to methane by anaerobic digestion: Co-effects of mixing intensity and long chain fatty acid loading

Oil in the food waste can contribute improved energy yield by anaerobic digestion, and also poses an inhibition risk from the intermediate long chain fatty acids (LCFAs). The digestion performance partly depends on the solubility of oil/LCFAs concerned recently, while mixing intensity control can disturb the solubility characteristics. Combined with oil loading such a fundamental control parameter, co-effects of mixing intensity and oil loading on methanogenic characteristics were illuminated. A series of mixing intensities 0–240 rpm and oil/inoculum (F/I) ratios 0.2–1.2 were set to conduct a batch experiment, and relevant affecting mechanisms were further disclosed as well. It turned out that variation of mixing intensity could have effects on methanogenesis from oil in food waste. Increasing mixing intensity prolonged lag phase. Mixing intensity at 120 rpm was proved as a starting point to disturb LCFA inhibition. Methanogenic rates climbed to the top at F/I 0.4, and further increase loading resulted in the rate decline. Methanogenic potential release was also reduced with both mixing intensity and loading at higher values. Hereinto, oleic acid (C18:1) as a key LCFA in oil played an important role in inhibition, while higher mixing intensity and loading could promote degradation and conversion of another critical LCFA palmitic acid (C16:0). The study favors achieving a cost-effective strategy controlled by mixing intensity to promote safe energy improvement.

Boosting anaerobic digestion of long chain fatty acid with microbial electrolysis cell combining metal organic framework as cathode: Biofilm construction and metabolic pathways

The role of metal organic framework (MOF) modified cathode in promoting long chain fatty acid (LCFA) methanation was identified in microbial electrolysis cell coupled anaerobic digestion (MEC-AD) system. The maximum methane production rate of MEC-AD-MOF achieved 49.8 ± 3.4 mL/d, which increased by 41 % compared to MEC-AD-C. The analysis of bio-cathode biofilm revealed that microbial activity, distribution, population, and protein secretion prompted by MOF cathode, which in turn led to an acceleration of electron transfer between the cathode and microbes. Specifically, the relative abundance of acetate-oxidizing bacterium (Mesotoga) in MEC-AD-MOF was 1.5–3.6 times higher than that in MEC-AD-C, with a co-metabolized enrichment of Methanobacterium. Moreover, MOF cathode reinforced LCFA methanation by raising the relative abundance of genes coded key enzymes involved in CO2-reducing pathway, and elevating the tolerance of microbes to LCFA inhibition. These results indicate that MOF can enhance biofilm construction in MEC-AD, thereby improving the treatment performance of lipid wastewater.

Revealing the synergy mechanisms of organic components anaerobic co-digestion from the prevailing tendency of endogenous inhibitors

The anaerobic co-digestion (AcoD) test among three organic components (model compounds) under high feeding load was carried out in this study, then the dynamic change of intermediate metabolic inhibitors was detected and analyzed to identify the interaction mechanism among organic components from the prevailing tendency of endogenous inhibitors. Carbohydrate and protein show significant two-way synergistic effects for methane production. The addition of protein can supplement additional alkalinity to alleviate the sharp drop of pH (from 4.56 to 6.73) and avoid the acidification caused by high concentration of carbohydrate. Additionally, volatile fatty acids (VFAs) produced by carbohydrate can transform free ammonia to ammonium ion with the largest drop of 54%, thus reducing ammonia toxicity. Carbohydrate and lipid show one-way synergistic effects, namely, lipid can reduce VFAs accumulation by 49% through long chain fatty acids (LCFAs) inhibiting action, and then avoid complete rancidity of carbohydrate fermentation at high concentration. However, protein and lipid show serious two-way antagonistic effects, especially under high load digestion. On one hand, LCFAs would weaken ammonia volatilization and thus aggravate ammonia inhibition; On the other hand, protein would not only accelerate the formation of LCFAs inhibition by promoting lipid hydrolysis, but also transform the degradation pathway of oleic acid (unsaturated LCFAs) and then increase the proportion of stearic acid from 5.17% to 40.33%, which is of higher toxicity than palmitic acid.

Comprehensive study of the combined effects of biochar and iron-based conductive materials on alleviating long chain fatty acids inhibition in anaerobic digestion

This study investigated the feasibility of alleviating the negative influence of long-chain fatty acids (LCFAs) on anaerobic digestion by biochar, micron zero-valent iron, micron-magnetite (mFe3O4) and their combination. The results demonstrate that co-addition of biochar and 6 g/L mFe3O4 (BC+6 g/L mFe3O4) increased cumulative methane production by 50% as suffered from LCFAs inhibition exerted by 2 g/L glycerol trioleate. The BC+6 g/L mFe3O4 did best in accelerating total organic carbon degradation and volatile fatty acids conversion, through successively enriching Bacteroides, Corynebacterium, and DMER64 to dominant the bacterial community. The proportion of acetotrophic Methanothrix that could alternatively reduce CO2 to methane by accepting electrons via direct interspecies electron transfer (DIET) was 0.09% with BC+6 g/L mFe3O4, nine times more than the proportion in control. Prediction of functional genes revealed the enrichment of the bacterial secretion system, indicating that BC+6 g/L mFe3O4 promoted DIET by stimulating the secretion of extracellular polymeric substances. This study provided novel insights into combining biochar and iron-based conductive materials to enhance AD performance under LCFAs inhibition.

Single bubble probe atomic force microscope and impinging-jet technique unravel the interfacial interactions controlled by long chain fatty acid in anaerobic digestion

Anaerobic digestion of lipid-rich wastewater generally suffers from foaming induced by long chain fatty acid (LCFA). However, a systematic understanding of LCFA inhibition, especially the physical inhibition on interfacial interaction still remains unclear. Here, we combined bubble probe atomic force microscope and impinging-jet technique to unravel the interfacial interactions controlled by long chain fatty acids in anaerobic digestion. We showed that LCFA had a significant inhibition on methane production in anaerobic reactors for the inhibition of the conversion of VFAs to methane. By measuring the LCFA influence on methanogenic archaea Methanosarcina acetivorans C2A, the results demonstrated that methanogenesis was limited for substrates utilization but not metabolic pathways. The impinging-jet technique results indicated that LCFA enhanced bubble separation from anaerobic granules and reduced the bubble-bubble coalescence probability. In addition, the bubble probe atomic force microscope (AFM) revealed that LCFA enhanced the adhesion force between bubbles by enhancing electrical double layer (EDL) repulsion and decreasing hydrophobic interactions. Overall, these results complement framework of LCFA inhibition in anerobic digestion and provide a nanomechanical insight into the fundamental interfacial interactions related to bubbles in anaerobic reactors.

The interaction between lipids and ammoniacal nitrogen mitigates inhibition in mesophilic anaerobic digestion

Ammoniacal nitrogen and long chain fatty acids (LCFA) are common inhibitors of the anaerobic digestion process. However, the interaction between these inhibitors has received little attention. Understanding the interaction between these inhibitors is important to optimise the operation of anaerobic digesters treating slaughterhouse waste or using fat, oil and grease (FOG) as co-substrate among others. To study the interaction between ammoniacal nitrogen and LCFA inhibition, 20 different conditions were trialled in mesophilic batch tests. Experimental conditions included 5 mixtures between slaughterhouse wastewater and LCFA (100:0, 75:25, 50:50, 20:80, 0:100 on a VS basis), each one tested at 4 different ammoniacal nitrogen concentrations (0, 1, 3, 6 gNadded·L-1). Experimental and modelling results showed that ammoniacal nitrogen inhibition was less severe in LCFA-rich mixtures, indicating that LCFA mitigated ammoniacal nitrogen inhibition to a certain extent. However, the positive interaction between inhibitors did not only depend on the LCFA concentration. A protective LCFA coat that limited the diffusion of free ammonia into the cell and/or provided a localised lower pH in the vicinity of the microbial cell could explain the experimental results. However, ammoniacal nitrogen and LCFA inhibition comprise up to 6 different but interrelated inhibitors (i.e. NH3, NH4+, LCFA, VFA, H2 and pH) and therefore the specific mechanism could not be elucidated. Nonetheless, these results suggest that LCFA do not exacerbate TAN-related inhibition and that LCFA-rich substrates can be utilised as co-substrates in mesophilic N-rich digesters.

Feeding strategies of continuous biomethanation processes during increasing organic loading with lipids or glucose for avoiding potential inhibition

Anaerobic co-digestion is a promising solution for nutrients balance and improvement of methane production in anaerobic digestion (AD) processes. However, the knowledge about the effects of different co-substrates in manure-based AD, and different feeding strategies, on the process performance and the methanogenic microbiome pathway, are still missing. Therefore, under harsh and slow stepwise increase of organic loading rate (OLR), by addition of lipids and carbohydrates as co-substrates in continuous reactors, this study elucidated their effect on methane production and methanogenic microbiome. The results showed that, when OLR increased by adding lipids, a severe inhibition due to accumulated long-chain fatty acids (LCFA) was observed, while no significant inhibition was obtained by addition of glucose. Additionally, the LCFA inhibition in the reactor fed with lipid was alleviated by slow stepwise feeding strategy that enriched aceticlastic Methanosarcina thermophile and Methanosaeta concilii, and hydrogenotrophic Methanobacterium methanogens.

Anaerobic co-digestion of municipal waste sludge with grease trap waste mixture: Point of process failure determination

Using high-strength wastes, such as grease trap waste mixture (GTWM) from multiple commercial facilities, as a co-substrate in anaerobic digestion can contribute to a significant increase in methane production. However, challenges such as long chain fatty acids (LCFAs) accumulation, sludge flotation, washout and scum formation can lead to digester failure. In this study, the optimal operating condition to achieve the highest methane production from an anaerobic, semi-continuous flow, mesophilic co-digester at a solids retention time of 20 days was determined. The ratio of GTWM/sludge was increased based on volatile solids (VS) concentration from 10% to 50% in 10% increments. A 40% GTWM/sludge ratio was determined to be optimum in which up to 22%, 200% and 68% improvements in VS removal, daily biogas production and specific methane production, respectively, were observed with respect to control utilizing sludge only. Beyond a 40% GTWM/sludge, the anaerobic co-digester experienced failure and addition of biochar to mitigate LCFA inhibition to failing co-digester could not recover the process. Digester LCFA concentrations were also measured during stable and unstable digester operations.

Glycerine supplementation as a recovery strategy of long-chain fatty acids inhibition on anaerobic digestion

In anaerobic digestion, less attention has been focused on the digestion of grease trap waste as a single substrate may be due to high lipid content that may cause inhibition effects resulted from long-chain fatty acids accumulation during the degradation of lipid. The inhibition results in the low and slow production of methane. This study investigates the influence of glycerine supplementation on the continuous stir tank reactor anaerobic treatment of grease trap waste. The operating procedures began with a start-up process and continued with a semicontinuous feeding process in an increasing organic loading rate of 1.3–3.6 gCOD/L day. The results showed that the resistance toward grease trap waste inhibition in a glycerine-supplemented reactor (RGS), measured as lag phase, can be shortened to 4 days during the start-up as compared to the control reactor (Rcontrol). With the reduction in lag-phase occurrence, the performance of RGS reactor affected the overall methane production. At standard temperature and pressure, RGS reactor performance of methane composition was 67%, with methane production rate of 0.376 LCH4/L day and methane yield of 0.19 LCH4/gCODremoved. These results were observed at an organic loading rate of 2.2 gCOD/L day. The overall methane production enhancement from the glycerine-supplemented reactor was 25%. Palmitate accumulation indicated the possible main inhibitor during the lag-phase experience. Furthermore, the system was able to recover methane production performance during semicontinuous feeding stage. The use of glycerine supplementation is a reliable strategy to induce the start-up of anaerobic digestion process of high-lipid substrate.

Mechanism, kinetics and microbiology of inhibition caused by long-chain fatty acids in anaerobic digestion of algal biomass.

BackgroundOleaginous microalgae contain a high level of lipids, which can be extracted and converted to biofuel. The lipid-extracted residue can then be further utilized through anaerobic digestion to produce biogas. However, long-chain fatty acids (LCFAs) have been identified as the main inhibitory factor on microbial activity of anaerobic consortium. In this study, the mechanism of LCFA inhibition on anaerobic digestion of whole and lipid-extracted algal biomass was investigated with a range of calcium concentrations against various inoculum to substrate ratios as a means to alleviate the LCFA inhibition.ResultsWhole algal biomass of Nannochloropsissalina represents high lipid content algal biomass while lipid-extracted residue represents its low lipid counterpart. The anaerobic digestion experiments were conducted in a series of serum bottles at 35 °C for 20 days. A kinetic model, considering LCFA inhibition on hydrolysis, acidogenesis as well as methanogenesis steps, was developed from the observed phenomenon of inhibition factors as a function of the LCFA concentration and specific biomass content or calcium concentration. The results showed that inoculum to substrate ratio had a stronger effect on biogas production than calcium, and calcium had no effect on biogas production when inoculum concentration was extremely low. The microbial community analysis by high-throughput Illumina Miseq sequencing indicated that diversity of both bacterial and methanogenic communities decreased with elevation of lipid concentration. Hydrolytic bacteria and aceticlastic methanogens dominated bacterial and archaea communities, respectively, in both high and low LCFA concentration digesters.ConclusionsThis study demonstrated that inoculum concentration has a more significant effect on alleviating LCFA inhibition than calcium concentration, while calcium only played a role when inoculum concentration met a threshold level. The model revealed that each functional microbial group was subject to different levels of LCFA inhibition. Although methanogens were the most susceptible microbes to LCFA inhibition, the inhibition factor for hydrolytic bacteria was more highly affected by inoculum concentration. The microbial community analysis indicated that the bacterial community was affected more than the methanogenic community by high LCFAs concentration. Syntrophic acetogens were sensitive to high LCFA concentrations and thus showed a decreased abundance in such an environment. Graphical abstractProposed mechanism of calcium mitigated LCFA inhibition

Dual-fuel production from restaurant grease trap waste: Bio-fuel oil extraction and anaerobic methane production from the post-extracted residue

An effective way for restaurant grease trap waste (GTW) treatment to generate fuel oil and methane by the combination of physiological and biological processes was investigated. The heat-driven extraction could provide a high purity oil equivalent to an A-grade fuel oil of Japanese industrial standard with 81–93wt% of extraction efficiency. A post-extracted residue was treated as an anaerobic digestion feedstock, and however, an inhibitory effect of long chain fatty acid (LCFA) was still a barrier for high-rate digestion. From the semi-continuous experiment fed with the residual sludge as a single substrate, it can be concluded that the continuous addition of calcium into the reactor contributed to reducing LCFA inhibition, resulting in the long-term stable operation over one year. Furthermore, the anaerobic reactor performed well with 70–80% of COD reduction and methane productivity under an organic loading rate up to 5.3g-COD/L/d.

Mesophilic co-digestion of dairy manure and lipid rich solid slaughterhouse wastes: Process efficiency, limitations and floating granules formation

Lipid and protein rich solid slaughterhouse wastes are attractive co-substrates to increase volumetric biogas production in co-digestion with dairy manure. Addition of decanter sludge (DS), containing 42.2% of lipids and 35.8% of proteins (total solids basis), up to 5% of feed mixture resulted in a stable process without any indication of long chain fatty acids (LCFA) or free ammonia (NH3) inhibition and in 3.5-fold increase of volumetric biogas production. Contrary, only lipids addition as technical fat (TF) at over 2% of feed mixture resulted in formation of floating granules (FG) and process efficiency decrease. Formed FG had low biodegradability and its organic part was composed of lipids and calcium salts of LCFAs. Anaerobic digestion process intentionally directed to FG formation, could be a viable option for mitigation and control of lipids overload and derived LCFA inhibition.

Postprandial inhibition of gastric ghrelin secretion by long-chain fatty acid through GPR120 in isolated gastric ghrelin cells and mice

Ghrelin is a gastric peptide hormone that controls appetite and energy homeostasis. Plasma ghrelin levels rise before a meal and fall quickly thereafter. Elucidation of the regulation of ghrelin secretion has been hampered by the difficulty of directly interrogating ghrelin cells diffusely scattered within the complex gastric mucosa. Therefore, we generated transgenic mice with ghrelin cell expression of green fluorescent protein (GFP) to enable characterization of ghrelin secretion in a pure population of isolated gastric ghrelin-expressing GFP (Ghr-GFP) cells. Using quantitative RT-PCR and immunofluorescence staining, we detected a high level of expression of the long-chain fatty acid (LCFA) receptor GPR120, while the other LCFA receptor, GPR40, was undetectable. In short-term-cultured pure Ghr-GFP cells, the LCFAs docosadienoic acid, linolenic acid, and palmitoleic acid significantly suppressed ghrelin secretion. The physiological mechanism of LCFA inhibition on ghrelin secretion was studied in mice. Serum ghrelin levels were transiently suppressed after gastric gavage of LCFA-rich lipid in mice with pylorus ligation, indicating that the ghrelin cell may directly sense increased gastric LCFA derived from ingested intraluminal lipids. Meal-induced increase in gastric mucosal LCFA was assessed by measuring the transcripts of markers for tissue uptake of LCFA, lipoprotein lipase (LPL), fatty acid translocase (CD36), glycosylphosphatidylinositol-anchored HDL-binding protein 1, and nuclear fatty acid receptor peroxisome proliferator-activated receptor-γ. Quantitative RT-PCR studies indicate significantly increased mRNA levels of lipoprotein lipase, glycosylphosphatidylinositol-anchored HDL-binding protein 1, and peroxisome proliferator-activated receptor-γ in postprandial gastric mucosa. These results suggest that meal-related increases in gastric mucosal LCFA interact with GPR120 on ghrelin cells to inhibit ghrelin secretion.

Long-chain fatty acids inhibition and adaptation process in anaerobic thermophilic digestion: Batch tests, microbial community structure and mathematical modelling

Biomass samples taken during the continuous operation of thermophilic anaerobic digestors fed with manure and exposed to successive inhibitory pulses of long-chain fatty acids (LCFA) were characterized in terms of specific metabolic activities and 16S rDNA DGGE profiling of the microbial community structure. Improvement of hydrogenotrophic and acidogenic (β-oxidation) activity rates was detected upon successive LCFA pulses, while different inhibition effects over specific anaerobic trophic groups were observed. Bioreactor recovery capacity and biomass adaptation to LCFA inhibition were verified. Population profiles of eubacterial and archaeal 16S rDNA genes revealed that no significant shift on microbial community composition took place upon biomass exposure to LCFA. DNA sequencing of predominant DGGE bands showed close phylogenetic affinity to ribotypes characteristic from specific β-oxidation bacterial genera ( Syntrophomonas and Clostridium), while a single predominant syntrophic archaeae was related with the genus Methanosarcina. The hypothesis that biomass adaptation was fundamentally of physiological nature was tested using mathematical modelling, taking the IWA ADM1 as general model. New kinetics considering the relation between LCFA inhibitory substrate concentration and specific biomass content, as an approximation to the adsorption process, improved the model fitting and provided a better insight on the physical nature of the LCFA inhibition process.

Strategies for recovering inhibition caused by long chain fatty acids on anaerobic thermophilic biogas reactors

Long chain fatty acids (LCFA) concentrations over 1.0 g L −1 were inhibiting manure thermophilic digestion, in batch and semi-continuous experiments, resulting in a temporary cease of the biogas production. The aim of the work was to test and evaluate several recovery actions, such as reactor feeding patterns, dilution and addition of adsorbents, in order to determine the most appropriate strategy for fast recovery of the reactor activity in manure based plants inhibited by LCFA. Dilution with active inoculum for increasing the biomass/LCFA ratio, or addition of adsorbents for adsorbing the LCFA and reducing the bioavailable LCFA concentration, were found to be the best recovery strategies, improving the recovery time from 10 to 2 days, in semi-continuously fed systems. Moreover, acclimatization was introduced by repeated inhibition and process recovery. The subsequent exposure of the anaerobic biomass to an inhibitory concentration of LCFA improved the recovery ability of the system, indicated as increasing degradation rates from 0.04 to 0.16 g COD_CH 4/g VS day. The incubation time between subsequent pulses, or discontinuous LCFA pulses, seems to be a decisive process parameter to tackle LCFA inhibition in manure anaerobic co-digestion.

Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes

Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with Type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A, leading to activation of glycogen synthase kinase 3. Phosphorylation and inactivation of glycogen synthase by glycogen synthase kinase 3 could be the mechanism for long-chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin-resistant subjects. Here, we test the effects of palmitic acid on cultured muscle glycogen synthase and PP2A activities. Palmitate inhibition of glycogen synthase fractional activity is increased in subjects with high body mass index compared with subjects with lower body mass index (r = -0.43, P = 0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2-h plasma glucose concentration to activation in subjects with increased 2-h plasma glucose concentration (r = 0.45, P < 0.03) during oral glucose tolerance tests. The results do not show an association between palmitate effects on PP2A and glycogen synthase fractional activity. We conclude that subjects at risk for Type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase), contributing to abnormal insulin regulation of glucose metabolism.

Inhibitory effects of long-chain fatty acids on VFA degradation and β-oxidation

The inhibitory effects of major long-chain fatty acids (LCFA), which have 16 or 18 carbons, not only on acetate degradation, but also on propionate degradation and beta-oxidation were examined in anaerobic serum bottle tests at 35 degrees C with the acclimated granular sludges. A modified Gompertz equation described cumulative methane production to assess the rates of VFA degradation and beta-oxidation, which were applied to a simplified noncompetitive model and a simplified substrate inhibition model, respectively. The specific methane production rates on acetate decreased as LCFA concentration increased, which was in good agreement with the noncompetitive inhibition model. Unsaturated oleate (C18:1) and linoleate (C18:2) were more inhibitory than saturated stearate (C18:0) and palmitate (C16:0) on acetate degradation. LCFA inhibition on propionate degradation was similar to that for acetate; however, propionate degradation was less inhibited than acetate degradation. Beta-oxidation was the rate-limiting step in LCFA degradation in most cases. As LCFA concentration increased, beta-oxidation rate reached the maximum value, and then decreased, which confirmed the substrate inhibition of LCFA. Oleate, the most abundant LCFA in wastewater, could be degraded more quickly than saturated LCFA containing the same or even less carbon in spite of relatively high toxicity on acetate degradation.

Abatement of long-chain fatty acid inhibition of methanogenesis by calcium addition

Long-chain fatty acids are versatile inhibitors for methanogenic bacteria. Research concerning lauric acid toxicity proved that addition of calcium chloride can reverse lauric acid inhibition of methanogenesis, provided the addition is applied within several minutes after the inhibitory lauric acid concentration has entered the methanogenic environment. The antagonistic effect of calcium chloride is caused by precipitation of the inhibitory long-chain fatty acids as calcium salts. It has been proved that lauric acid has a strong affinity for methanogenic sludge. The rapid reaction of lauric acid with methanogenic sludge causes the need for almost immediate addition of calcium chloride to prevent inhibition of methanogenesis. Of the methanogenic potential of granular sludge 50% was lost after only 7·5 min of exposure. Addition of calcium chloride after an exposure period of 6 h had no effect at all.

Long-chain fatty acid inhibition of growth of Streptococcus agalactiae in a chemically defined medium.

Willett, Norman P. (University of Pennsylvania School of Veterinary Medicine, Kennett Square, Pa.), and Guy E. Morse. Long-chain fatty acid inhibition of growth of Streptococcus agalactiae in a chemically defined medium. J. Bacteriol. 91:2245-2250. 1966.-A chemically defined medium was developed for Streptococcus agalactiae which supported growth comparable to that obtained in complex medium. The effects of long-chain fatty acids on growth of the organisms were determined turbidimetrically. The order of activity of the fatty acids was dependent upon whether complete inhibition or median response (50% inhibition point) was used as a parameter of activity. When complete inhibition of growth was used as a measure, the degree of unsaturation of C(18) acids enhanced antimicrobial activity. However, when the median response was used as an index, this order was reversed. Increase in carbon chain from C(12) to C(18) did not correlate with either complete inhibition or median response points. Antimicrobial activity of unsaturated and saturated fatty acids was reversed by bovine serum albumin and other compounds, suggesting a bacteriostatic action.