Large volumes of untreated palm oil mill effluent (POME) pose threat to aquatic environment due to the presence of very high organic content. The present investigation involved two pilot-scale anaerobic expanded granular sludge bed (EGSB) reactors, continuously operated for 1 year to treat POME. Setting HRT at 9.8 d, the anaerobic EGSB reactors reduced COD from 71179 mg/L to 12341 mg/L and recycled half of sludge by a dissolved air flotation (DAF). The average effluent COD was 3587 mg/L with the consistent COD removal efficiency of 94.89%. Adding cationic polymer (PAM) dose of 30 mg/L to DAF unit and recycling its half of sludge caused granulation of anaerobic sludge. Bacilli and small coccid bacteria were the dominant microbial species of the reactor. The reactor produced 27.65 m(3) of biogas per m(3) of POME which was utilized for electricity generation.
Mercuric species, Hg(II), interacts strongly with dissolved organic matter (DOM) through the oxidation, reduction, and complexation that affect the fate, bioavailability, and cycling of mercury, Hg, in aquatic environments. Despite its importance, the reactions between Hg(II) and DOM have rarely been studied in the presence of different concentrations of chloride ions (Cl-) under anoxic conditions. Here, we report that the extent of Hg(II) reduction in the presence of the reduced DOM decreases with increasing Cl- concentrations. The rate constants of Hg(II) reduction ranged from 0.14 to 1.73 h-1 in the presence of Cl- and were lower than the rate constant (2.41 h-1) in the absence of Cl-. Using a thermodynamic model, we showed that stable Hg(II)-chloride complexes were formed in the presence of Cl-. We further examined that H(0) was oxidized to Hg(II) in the presence of the reduced DOM and Cl- under anoxic conditions, indicating that Hg(II) reduction is inhibited by the Hg(0) oxidation. Therefore, the Hg(II) reduction by the reduced DOM can be offset due to the Hg(II)-chloride complexation and Hg(0) oxidation in chloride-rich environments. These processes can significantly influence the speciation of Hg and have an important implication for the behavior of Hg under environmentally relevant concentrations.
Pandoraea species have been isolated from diverse environmental samples and are emerging important respiratory pathogens, particularly in people with cystic fibrosis (CF). In the present study, two bacterial isolates initially recovered from consecutive sputum samples collected from a CF patient and identified as Pandoraea pnomenusa underwent a polyphasic taxonomic analysis. The isolates were found to be Gram-negative, facultative anaerobic motile bacilli and subsequently designated as strains 6399T (=LMG29626T=DSM103228T) and 7641 (=LMG29627=DSM103229), respectively. Phylogenetic analysis based on 16S rRNA and gyrB gene sequences revealed that 6399T and 7641 formed a distinct phylogenetic lineage within the genus Pandoraea. Genome sequence comparison analysis indicated that strains 6399T and 7641 are clonal and share 100 % similarity, however, similarity to other type strains (ANIb 73.2-88.8 %, ANIm 83.5-89.9 % and OrthoANI 83.2-89.3 %) indicates that 6399T and 7641 do not belong to any of the reported type species. The major cellular fatty acids of 6399T were C16 : 0 (32.1 %) C17 : 0cyclo (18.7 %) and C18 : 1ω7c (14.5 %), while Q-8 was the only respiratory quinone detected. The major polar lipids identified were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The genomic DNA G+C content of 6399T was 62.9 (mol%). Strain 6399T can be differentiated from other members of Pandoraea by the absence of C19 : 0ω8c cyclo and by the presence of C17 : 0ω8c cyclo. Together our data show that the bacterial strains 6399T and 7641 represent a novel species of the genus Pandoraea, for which the name Pandoraea fibrosis sp. nov. is proposed (type strain 6399T).
Empty fruit bunch (EFB) and palm oil mill effluent (POME) are the major wastes generated by the oil palm industry in Malaysia. The practice of EFB and POME digester sludge co-composting has shown positive results, both in mitigating otherwise environmentally damaging waste streams and producing a useful product (compost) from these streams. In this study, the bacterial ecosystems of 12-week-old EFB-POME co-compost and POME biogas sludge from Felda Maokil, Johor were analysed using 16S metagenome sequencing. Over ten phyla were detected, with Chloroflexi being the predominant phylum, representing approximately 53% of compost and 23% of the POME microbiome reads. The main bacterial lineage found in the compost and POME was Anaerolinaceae (Chloroflexi) with 30% and 18% of the total gene fragments, respectively. The significant differences between compost and POME communities were abundances of Syntrophobacter, Sulfuricurvum and Coprococcus. No methanogens were identified due to the bias in general 16S primers to eubacteria. The preponderance of anaerobic species in the compost and high abundance of secondary metabolite fermenting bacteria is due to an extended composting time, with anaerobic collapse of the pile due to the tropical heat. Predictive functional profiles of the metagenomes using 16S rRNA marker genes suggest that the presence of enzymes involved in degradation of polysaccharides such as glucoamylase, endoglucanase and arabinofuranosidase, all of which were strongly active in POME. Eubacterial species associated with cellulytic methanogenesis were present in both samples.
The intestinal microbiota is increasingly recognized as an important component of host health, metabolism and immunity. Early gut colonizers are pivotal in the establishment of microbial community structures affecting the health and growth performance of chickens. White Lohmann layer is a common commercial breed. Therefore, this breed was selected to study the pattern of changes of microbiota with age. In this study, the duodenum, caecum and colorectum contents of white Lohmann layer chickens from same environment control farm were collected and analyzed using 16S rRNA sequencing to explore the spatial and temporal variations in intestinal microbiota. The results showed that the diversity of the microbial community structure in the duodenum, caecum and colorectum increased with age and tended to be stable when the layer chickens reached 50 days of age and the distinct succession patterns of the intestinal microbiota between the duodenum and large intestine (caecum and colorectum). On day 0, the diversity of microbes in the duodenum was higher than that in the caecum and colorectum, but the compositions of intestinal microbes were relatively similar, with facultative anaerobic Proteobacteria as the main microbes. However, the relative abundance of facultative anaerobic bacteria (Escherichia) gradually decreased and was replaced by anaerobic bacteria (Bacteroides and Ruminococcaceae). By day 50, the structure of intestinal microbes had gradually become stable, and Lactobacillus was the dominant bacteria in the duodenum (41.1%). The compositions of dominant microbes in the caecum and colorectum were more complex, but there were certain similarities. Bacteroides, Odoribacter and Clostridiales vadin BB60 group were dominant. The results of this study provide evidence that time and spatial factors are important factors affecting the intestinal microbiota composition. This study provides new knowledge of the intestinal microbiota colonization pattern of layer chickens in early life to improve the intestinal health of layer chickens.
Anaerobic digestion is a process by which microorganisms break down biodegradable material in the absence of oxygen. The process involves hydrolysis, acidogenesis and methanogenesis stages. Anaerobic digestion of food waste has been widely investigated for biogas recovery but limited study was performed on phosphorus recovery, which is reported depleting. Food waste is produced every day and dumped on landfill for final disposal which may lead to environmental issues such as odour problems and greenhouse gases release, due to decomposing of food waste, hence impacts global climate change. In anaerobic digestion pH is a very crucial parameter in an attempt to recover phosphorus as it highly influences the production of organic acids during acidogenesis.
The present work aimed to determine the antagonistic effect of probiotic-fermented soy against oral pathogens. Indigenous oral probiotics (Streptococcus salivarius Taylor’s Univer- sity Collection Centre (TUCC) 1251, S. salivarius TUCC 1253, S. salivarius TUCC 1254, S. salivarius TUCC 1255, and S. orisratti TUCC 1253) were incorporated into soy fermentation at 37°C for 24 h. Growth characteristics, β-glucosidase activity, and total isoflavones content of Streptococcus strains following soy fermentation were analysed. Antimicrobial test of Streptococcus-fermented soy was carried out against oral pathogens Enterococcus faecalis American Type Culture Collection (ATCC) 700802, Streptococcus pyogenes ATCC 19615, and Staphylococcus aureus ATCC 25923. Streptococcus strains showed a significant increase in growth following soy fermentation. S. salivarius TUCC 1253-fermented soy showed signif- icantly higher extracellular β-glucosidase activity and amount of aglycones. S. salivarius TUCC 1253-fermented soy showed antimicrobial effect against all oral tested pathogens in both aerobic and anaerobic conditions. These results showed that S. salivarius TUCC 1253-fermented soy could potentially be used as a preventive action or alternative treatment for oral infections.
Thriving oil palm agroindustry comes at a price of voluminous waste generation, with palm oil mill effluent (POME) as the most cumbersome waste due to its liquid state, high strength, and great discharge volume. In view of incompetent conventional ponding treatment, a voluminous number of publications on non-conventional POME treatments is filed in the Scopus database, mainly working on alternative or polishing POME treatments. In dearth of such comprehensive review, all the non-conventional POME treatments are rigorously reviewed in a conceptual and comparative manner. Herein, non-conventional POME treatments are sorted into the five major routes, viz. biological (bioconversions - aerobic/anaerobic biodegradation), physical (flotation & membrane filtration), chemical (Fenton oxidation), physicochemical (photooxidation, steam reforming, coagulation-flocculation, adsorption, & ultrasonication), and bioelectrochemical (microbial fuel cell) pathways. For aforementioned treatments, the constraints, pros, and cons are qualitatively and quantitatively (with compiled performance data) detailed to indicate their process maturity. Authors recommended (i) bioconversions, adsorption, and steam reforming as primary treatments, (ii) flotation and ultrasonication as pretreatments, (iii) Fenton oxidation, photooxidation, and membrane filtration as polishing treatments, and (iv) microbial fuel cell and coagulation-flocculation as pretreatment or polishing treatment. Life cycle assessments are required to evaluate the environmental, economic, and energy aspects of each process.
As the population increases, energy demands continue to rise rapidly. In order to satisfy this increasing energy demand, biogas offers a potential alternative. Biogas is economically viable to be produced through anaerobic digestion (AD) from various biomass feedstocks that are readily available in Malaysia, such as food waste (FW), palm oil mill effluent (POME), garden waste (GW), landfill, sewage sludge (SS) and animal manure. This paper aims to determine the potential feedstocks for biogas production via AD based on their characteristics, methane yield, kinetic studies and economic analysis. POME and FW show the highest methane yield with biogas yields up to 0.50 L/g VS while the lowest is 0.12 L/g VS by landfill leachate. Kinetic study shows that modified Gompertz model fits most of the feedstock with R 2 up to 1 indicating that this model can be used for estimating treatment efficiencies of full-scale reactors and performing scale-up analysis. The economic analysis shows that POME has the shortest payback period (PBP), highest internal rate of return (IRR) and net present value (NPV). However, it has already been well explored, with 93% of biogas plants in Malaysia using POME as feedstock. The FW generation rate in Malaysia is approximately 15,000 tonnes per day, at the same time FW as the second place shows potential to have a PBP of 5.4 years and 13.3% IRR, which is close to the results achieved with POME. This makes FW suitable to be used as the feedstock for biogas production.
The performance of modified anaerobic inclining-baffled reactor (MAI-BR) treating recycled paper mill effluent (RPME) was investigated by varying the influent chemical oxygen demand (CODin) concentration from 1000 to 4000 mg/L, and the hydraulic retention time (HRT) from 3 to 1 day, corresponding to an organic loading rate increase from 0.33 to 4 g COD/L day. Throughout 126 days of operation, a maximum removal efficiency of up to 96% of chemical oxygen demand (COD) and 99% of biological oxygen demand, methane (CH4) yield of 0.259 L CH4/g COD, and a stable effluent pH of 6.5 were achieved. Furthermore, the compartmental performance showed that most of the organic substrates were removed in the initial two compartments, resulting in low pH and alkalinity levels and a high concentration of volatile fatty acids. Overall, the results showed that the MAI-BR successfully treated RPME, and the performance was affected by the variation of HRT more than the CODin.
In this study, the performance of a laboratory scale upflow anaerobic sludge blanket (UASB) reactor operating at mesophilic temperature (35 °C) was examined. Cattle slaughterhouse wastewater (CSWW) was used as the main substrate. The total and effective volumes of the reactor were 8 L and 6 L, respectively. Twelve different organic loading rates (OLR) were applied and the performance was evaluated. The chemical oxygen demand (COD) removal efficiency was more than 90% during batch study. In the continuous study, COD removal was also approximately 90% at OLR 0.4 g/L d-1 which subsequently dropped to below 50% when the loading rate increased to 15 g/L d-1. Approximately 5 L/d of biogas was obtained with high methane concentration at stages VI and XI corresponding to OLR of 2 and 10 g/L d-1, respectively. It was observed that the concentration of volatile fatty acids was low and that the alkalinity of the wastewater was sufficient to avoid acidification. Specific methane yields of 0.36 and 0.38 LCH₄/g COD added were achieved at OLR 7 and 10 g/L d-1. A hydraulic retention time (HRT) of 1 day was sufficient to remove greater than 70% of COD which correspond to 89% methane concentration. Parameters like soluble COD, NH₃-N, pH, alkalinity, total suspended solid (TSS), fats, oil, and grease were also investigated. The results show that the UASB reactor could serve as a good alternative for anaerobic treatment of CSWW and methane production.
Complete degradation of azo dye has always been a challenge due to the refractory nature of azo dye. An innovative hybrid system, constructed wetland-microbial fuel cell (CW-MFC) was developed for simultaneous azo dye remediation and energy recovery. This study investigated the effect of circuit connection and the influence of azo dye molecular structures on the degradation rate of azo dye and bioelectricity generation. The closed circuit system exhibited higher chemical oxygen demand (COD) removal and decolourisation efficiencies compared to the open circuit system. The wastewater treatment performances of different operating systems were ranked in the decreasing order of CW-MFC (R1 planted-closed circuit) > MFC (R2 plant-free-closed circuit) > CW (R1 planted-open circuit) > bioreactor (R2 plant-free-open circuit). The highest decolourisation rate was achieved by Acid Red 18 (AR18), 96%, followed by Acid Orange 7 (AO7), 67% and Congo Red (CR), 60%. The voltage outputs of the three azo dyes were ranked in the decreasing order of AR18 > AO7 > CR. The results disclosed that the decolourisation performance was significantly influenced by the azo dye structure and the moieties at the proximity of azo bond; the naphthol type azo dye with a lower number of azo bond and more electron-withdrawing groups could cause azo bond to be more electrophilic and more reductive for decolourisation. Moreover, the degradation pathway of AR18, AO7 and CR were elucidated based on the respective dye intermediate products identified through UV-Vis spectrophotometry, high-performance liquid chromatography (HPLC), and gas chromatograph-mass spectrometer (GC-MS) analyses. The CW-MFC system demonstrated high capability of decolouring azo dyes at the anaerobic anodic region and further mineralising dye intermediates at the aerobic cathodic region to less harmful or non-toxic products.
In this study, a two-stage domesticated shear-loop anaerobic contact stabilization (SLACS) system is introduced as a new reactor design to enhance methane productivity with significant reduction in hydrogen sulphide (H2S) synthesis. Due to the rich sulfate content in industrial wastewaters, the initial fermentation phase of anaerobic digestion is highly acidifying and often leads to severe performance losses, digester's instability, and even culture crash. The SLACS system functions as a dissimilatory sulfate reduction - methanogenic reactor consisting of two compartments, a shear-loop anaerobic bed (SLAB) unit and an anaerobic plug flow (APF) unit. The functional role of the SLAB unit is not limited to acidogenesis but also sulfidogenic processes, which curtails H2S generation in the APF unit (methanogenic stage). Experimental observations indicated that pH serves a critical role in the cohabitation of acidogenic and sulfidogenic microbes in the SLAB unit. Although acidogenesis was not influenced by pH within the range of 4.5-6.0, it is vital to stabilize the pH of this unit at 5.4 to establish a steady sulfate reduction of above 75%. The highest desulfurization achieved in this compartment was 88% under a hydraulic retention time (HRT) of 4 h. With an average methane productivity of 256 mL g-1 VS, the methanogenic performance of the two-stage domesticated SLACS system shows a 32% methanogenic proficiency higher than that of the one-stage digestion system. Microbial community structure within the system carried out via Next Generation Sequencing (NGS) provided qualitative data on the sludge's sulfidogenic and methanogenic performance.
Three different sizes of powdered activated carbon (PAC) were added in hybrid anaerobic membrane bioreactors (AnMBRs) and their performance was compared with a conventional AnMBR without PAC in treating palm oil mill effluent. Their working volume was 1 L each. From the result, AnMBRs with PAC performed better than the AnMBR without PAC. It was also found that adding a relatively smaller size of PAC (approximately 100 μm) enhanced the chemical oxygen demand removal efficiency to 78.53 ± 0.66%, while the concentration of mixed liquor suspended solid and mixed liquor volatile suspended solid were 8,050 and 6,850 mg/L, respectively. The smaller size of PAC could also enhance the biofloc formation and biogas production. In addition, the smaller particle sizes of PAC incorporated into polyethersulfone membrane resulted in higher performance of membrane fouling control and produced better quality of effluent as compared to the membrane without the addition of PAC.
Malaysian authorities has planned to minimize and stop when applicable unsanitary dumping of waste as it puts human health and the environment at elevated risk. Cost, energy and revenue are mostly adopted to draw the blueprint of upgrading municipal solid waste management system, while the carbon footprint emissions criterion rarely acts asa crucial factor. This study aims to alert Malaysian stakeholders on the uneven danger of carbon footprint emissions of waste technologies. Hence, three scenarios have been proposed and assessed mainly on the carbon footprint emissions using the 2006 IPCC methodology. The first scenario is waste dumping in sanitary landfills equipped with gas recovery system, while the second scenario includes anaerobic digestion of organics and recycling of recyclable wastes such as plastic, glass and textile wastes. The third scenario is waste incineration. Besides the carbon footprint emissions criterion, other environmental concerns were also examined. The results showed that the second scenario recorded the lowest carbon footprint emissions of 0.251t CO2 eq./t MSW while the third scenario had the highest emissions of 0.646t CO2 eq./t MSW. Additionally, the integration between anaerobic digestion and recycling techniques caused the highest avoided CO2 eq. emissions of 0.74t CO2 eq./t MSW. The net CO2 eq. emissions of the second scenario equaled -0.489t CO2 eq./t MSW due to energy recovery from the biogas and because of recycled plastic, glass and textile wastes that could replace usage of raw material. The outcomes also showed that the first scenario generates huge amount of leachate and hazardous air constituents. The study estimated that a ton of dumped waste inside the landfills generates approximately 0.88m3 of trace risky compounds and 0.188m3 of leachate. As for energy production, the results showed that the third scenario is capable of generating 639kWh/t MSW followed by the second scenario with 387.59kWh/t MSW. The first scenario produced 296.79kWh/t MSW. In conclusion, the outcomes of this study recommend an integrated scenario of anaerobic digestion and recycling techniques to be employed in Malaysia.
Treatment of ammonia- and nitrate-rich wastewater, such as that generated in the aquaculture industry, is important to prevent environmental pollution. The anaerobic ammonium oxidation (anammox) process has been reported as a great alternative in reducing ammoniacal nitrogen concentration in aquaculture wastewater treatment compared to conventional treatment systems. This paper will highlight the impact of the anammox process on aquaculture wastewater, particularly in the regulation of ammonia and nitrogen compounds. The state of the art for anammox treatment systems is discussed in comparison to other available treatment methods. While the anammox process is viable for the treatment of aquaculture wastewater, the efficiency of nitrogen removal could be further improved through the proper use of anammox bacteria, operating conditions, and microbial diversity. In conclusion, a new model of the anammox process is proposed in this review.
In this study, the treatment of poultry slaughterhouse wastewater (PSW) was evaluated using two new down-flow high-rate anaerobic bioreactor systems (HRABS), including the down-flow expanded granular bed reactor (DEGBR) and the static granular bed reactor (SGBR). These two bioreactors have demonstrated a good performance for the treatment of PSW with removal percentages of the biochemical oxygen demand (BOD5), the chemical oxygen demand (COD), and fats, oil, and grease (FOG) exceeding 95% during peak performance days. This performance of down-flow HRABS appears as a breakthrough in the field of anaerobic treatment of medium to high-strength wastewater because down-flow anaerobic bioreactors have been neglected for the high-rate anaerobic treatment of such wastewater due to the success of up-flow anaerobic reactors such as the UASB and the EGSB as a result of the granulation of a consortium of anaerobic bacteria required for efficient anaerobic digestion and biogas production. Hence, to promote the recourse to such technologies and provide further explanation to their performance, this study approached the kinetic analysis of these two down-flow HRABS using the modified Stover-Kincannon and the Grau second-order multi-component substrate models. From a comparison between the two models investigated, the modified Stover-Kincannon model provided the best prediction for the concentration of the substrate in the effluent from the two HRABS. This analysis led to the determination of the kinetic parameters of the two models that can be used for the design of the two HRABS and the prediction of the performance of the SGBR and DEGBR. The kinetic parameters determined using the Modified Stover-Kincannon were Umax = 40.5 gCOD/L.day and KB = 47.3 gCOD/L.day for the DEGBR and Umax = 33.6 gCOD/L.day and KB = 44.9 gCOD/L.day for the SGBR; while, using the Grau second-order model, the kinetic models determined were a = 0.058 and b = 1.112 for the DEGBR and a = 0.135 and b = 1.33 for the SGBR.
Cattle slaughterhouses generate wastewater that is rich in organic contaminant and nutrients, which is considered as high strength wastewater with a high potential for energy recovery. Work was undertaken to evaluate the efficiency of the 12 L laboratory scale conventional and a modified upflow anaerobic sludge blanket (UASB) reactors (conventional, R1 and modified, R2), for treatment of cattle slaughterhouse wastewater (CSWW) under mesophilic condition (35 ± 1 °C). Both reactors were acclimated with synthetic wastewater for 30 days, then continuous study with real CSWW proceeds. The reactors were subjected to the same loading condition of OLR, starting from 1.75, 3, 5 10, 14, and 16 g L-1d-1, corresponding to 3.5, 6, 10, 20, 28, and 32 g COD/L at constant hydraulic retention time (HRT) of 24 h. The performance of the R1 reactor drastically dropped at OLR 10 g L-1d-1, and this significantly affected the subsequent stages. The steady-state performance of the R2 reactor under the same loading condition as the R1 reactor revealed a high COD removal efficiency of 94% and biogas and methane productions were 27 L/d and 89%. The SMP was 0.21 LCH4/gCOD added, whereas the NH3-N alkalinity ratio stood at 651 mg/L and 0.2. SEM showed that the R2 reactor was dominated by Methanosarcina bacterial species, while the R1 reactor revealed a disturb sludge with insufficient microbial biomass.
Anaerobic co-digestion (co-AD) of agro-industrial waste, namely, palm oil mill effluent (POME) and sugarcane vinasse (Vn), with water hyacinth (WH) as co-substrate was carried out in two separate Anaerobic Suspended Growth Closed Bioreactors (ASGCBs) under thermophilic (55 °C) conditions. The highest chemical oxygen demand (COD) and soluble COD reduction in co-AD of POME-WH (78.61%, 78.86%) is slightly higher than co-AD of Vn-WH (75.75%, 78.24%). However, VFA reduction in co-AD of POME-WH (96.41%) is higher compared to co-AD of Vn-WH (85.94%). Subsequently, biogas production peaked at 13438 mL/day values and 16122 mL/day for co-AD of POME-WH and Vn-WH, respectively. However, the methane content was higher in the co-AD of POME-WH (72.04%) than in the co-AD of Vn-WH (69.86%). Growth yield (YG), maximum specific substrate utilization rate (rx,max) and maximum specific biomass growth rate (μmax) are higher in co-AD of POME-WH, as supported by the higher mixed liquor volatile suspended solids (MLVSS) and COD reduction efficiency compared to co-AD of Vn-WH. However, methane yield ([Formula: see text]) reported in the co-AD of POME-WH and Vn-WH are 0.2748 and 0.3112 L CH4/g CODreduction, respectively, which suggests that WH is a more suitable co-substrate for Vn compared to POME.
A Mo -reducing bacterium (strain 48), which grew on medium supplemented with 200 mM Mo, was isolated from stream water obtained from Chengkau, Malaysia. The chemical properties of strain 48 conform to the characteristics of Enterobacter cloacae. Under anaerobic conditions in the glucose-yeast extract medium containing phosphate ion (2.9 mM) and Mo (10 mM), the bacterium reduced Mo to form molybdenum blue. Approximately 27% of Mo added to the medium was reduced after 28 h of cultivation. The reduction of Mo with glucose as an electron donor was strongly inhibited by iodoacetic acid, sodium fluoride, and sodium cyanide, suggesting an involvement of the glycolytic pathway and electron transport in Mo reduction. NADH and N,N,N',N' -tetramethyl-p-phenylenediamine served as electron donors for Mo reduction. When NADH was used as an electron donor, at first cytochrome b in the cell extract was reduced, and then molybdenum blue was formed. Sodium cyanide strongly inhibited Mo reduction by NADH (5 mM) but not the reduction of cytochrome b in the cell extract, suggesting that the reduced component of the electron transport system after cytochrome b serves as an electron donor for Mo reduction. Both ferric and stannous ions strongly enhanced the activity of Mo reduction by NADH.