Biological Control of Hog Waste Odor through Stimulated Microbial Fe(III) Reduction

Odor control and disposal of swine waste have inhibited expansion of swine production facilities throughout the United States. Swine waste odor is associated primarily with high concentrations of volatile fatty acids (VFAs). Here, we demonstrate that stimulated Fe(III) reduction in hog manure can rapidly remove the malodorous compounds and enhance methane production by 200%. As part of these studies, we enumerated the indigenous Fe(III)-reducing population in swine waste and identified members of the family Geobacteraceae as the dominant species. These organisms were present at concentrations as high as 2 × 10⁵ cells g⁻¹. Several pure cultures of Fe(III) reducers, including Geobacter metallireducens, Geobacter humireducens, Geobacter sulfurreducens, Geobacter grbiciae, Geothrix fermentans, and Geovibrio ferrireducens, readily degraded some or all of the malodorous VFAs found in swine manure. In contrast, Shewanella algae did not degrade any of these compounds. We isolated an Fe(III) reducer, Geobacter strain NU, from materials collected from primary swine waste lagoons. This organism degraded all of the malodorous VFAs tested and readily grew in swine waste amended with Fe(III). When raw waste amended with Fe(III) was inoculated with strain NU, the VFA content rapidly decreased, corresponding with an almost complete removal of the odor. In contrast, the raw waste without Fe(III) or strain NU showed a marked increase in VFA content and a rapid pH drop. This study showed that Fe(III) supplementation combined with appropriate bioaugmentation provides a simple, cost-effective approach to deodorize and treat swine waste, removing a significant impediment to the expansion of pork production facilities.     

Biological control of hog waste odor through stimulated microbial Fe(III) reduction

Odor control and disposal of swine waste have inhibited expansion of swine production facilities throughout the United States. Swine waste odor is associated primarily with high concentrations of volatile fatty acids (VFAs). Here, we demonstrate that stimulated Fe(III) reduction in hog manure can rapidly remove the malodorous compounds and enhance methane production by 200%. As part of these studies, we enumerated the indigenous Fe(III)-reducing population in swine waste and identified members of the family Geobacteraceae as the dominant species. These organisms were present at concentrations as high as 2 x 10(5) cells g(-1). Several pure cultures of Fe(III) reducers, including Geobacter metallireducens, Geobacter humireducens, Geobacter sulfurreducens, Geobacter grbiciae, Geothrix fermentans, and Geovibrio ferrireducens, readily degraded some or all of the malodorous VFAs found in swine manure. In contrast, Shewanella algae did not degrade any of these compounds. We isolated an Fe(III) reducer, Geobacter strain NU, from materials collected from primary swine waste lagoons. This organism degraded all of the malodorous VFAs tested and readily grew in swine waste amended with Fe(III). When raw waste amended with Fe(III) was inoculated with strain NU, the VFA content rapidly decreased, corresponding with an almost complete removal of the odor. In contrast, the raw waste without Fe(III) or strain NU showed a marked increase in VFA content and a rapid pH drop. This study showed that Fe(III) supplementation combined with appropriate bioaugmentation provides a simple, cost-effective approach to deodorize and treat swine waste, removing a significant impediment to the expansion of pork production facilities.     

Influence of Naturally Occurring Humic Acids on Biodegradation of Monosubstituted Phenols by Aquatic Bacteria

Samples of the microbial community from Lake Michie, a mesotrophic reservoir in central North Carolina, were adapted to various levels (100 to 1,000 μg/liter) of natural humic acids in chemostats. The humic acids were extracted from water samples from Black Lake, a highly colored lake in the coastal plain of North Carolina. After adaptation, the microbial community was tested for its ability to degrade the monosubstituted phenols m-cresol, m-aminophenol, and p-chlorophenol. Adaptation to increasing levels of humic acids significantly reduced the ability of the microbial communities to degrade all three phenols. The decline in biodegradation was accompanied by a decrease in the number of specific compound degraders in the adapted communities. Short-term exposure of the community to increasing levels of humic acids had no significant effect on the ability of the community to degrade m-cresol. Thus the suppressive effect of humic acids on monosubstituted phenol metabolism was the result of long-term exposure to the humic materials. Increasing the levels of inorganic nutrients fed to the chemostats during the humic acid adaptation had little effect on the suppressive influence of the humic acids, indicating that nutrient limitation was probably not responsible for the metabolic suppression. The results of the study suggest that long-term exposure to humic acids can reduce the ability of microbial communities to respond to monosubstituted phenols.     

Degradation of Tyrosine in Anaerobically Stored Piggery Wastes and in Pig Feces

Radioactively labeled compounds that might be intermediates in the anaerobic degradation of tyrosine were added to pig feces and to stored piggery wastes. Changes in the compounds were followed by using thin-layer and gas chromatography. In feces, p-cresol and 3-phenylpropionic acid were the end products of tyrosine metabolism; in anaerobically stored mixed wastes, phenol, p-cresol, and minor quantities of phenylpropionic acid were formed. Schemes were proposed for the degradation of tyrosine in pig feces and in mixed wastes.     

Key enzymes of the protocatechuate branch of the β-ketoadipate pathway for aromatic degradation inCorynebacterium glutamicum

Although the protocatechuate branch of the β-ketoadipate pathway in Gram⁻ bacteria has been well studied, this branch is less understood in Gram⁺ bacteria. In this study,Corynebacterium glutamicum was cultivated with protocatechuate,p-cresol, vanillate and 4-hydroxybenzoate as sole carbon and energy sources for growth. Enzymatic assays indicated that growing cells on these aromatic compounds exhibited protocatechuate 3,4-dioxygenase activities. Data-mining of the genome of this bacterium revealed that the genetic locusncg12314-ncg12315 encoded a putative protocatechuate 3,4-dioxygenase. The genes,ncg12314 andncg12315, were amplified by PCR technique and were cloned into plasmid (pET21aP34D). RecombinantEscherichia coli strain harboring this plasmid actively expressed protocatechuate 3,4-dioxygenase activity. Further, when this locus was disrupted inC. glutamicum, the ability to degrade and assimilate protocatechuate,p-cresol, vanillate or 4-hydroxybenzoate was lost and protocatechuate 3,4-dioxygenase activity was disappeared. The ability to grow with these aromatic compounds and protocatechuate 3,4-dioxygenase activity ofC. glutamicum mutant could be restored by gene complementation. Thus, it is clear that the key enzyme for ring-cleavage, protocatechuate 3,4-dioxygenase, was encoded byncg12314 andncg12315. The additional genes involved in the protocatechuate branch of the β-ketoadipate pathway were identified by mining the genome data publically available in the Gen Bank. The functional identification of genes and their unique organization inC. glutamicum provided new insight into the genetic diversity of aromatic compound degradation.     

Eugenol stimulates lactate accumulation yet inhibits volatile fatty acid production and eliminates coliform bacteria in cattle and swine waste

AIM: To determine how eugenol affects fermentation parameters and faecal coliforms in cattle and swine waste slurries stored anaerobically. METHODS AND RESULTS: Waste slurries (faeces:urine:water, 50:35:15) were blended with and without additives and aliquoted to triplicate 1-l flasks. Faecal coliforms were eliminated in cattle and swine waste slurries within 1 or 2 days with additions of eugenol at 10.05 mm (0.15%) and 16.75 mm (0.25%). At these concentrations volatile fatty acids (VFA) were reduced ca 70 and 50% in cattle and swine waste, respectively, over 6-8 weeks. Additionally, in cattle waste, eugenol stimulated the accumulation of lactate (>180 mm) when compared with thymol treatment (20 mm lactate). In swine waste, lactate accumulation did not occur without additives; eugenol and thymol stimulated lactate accumulation to concentrations of 22 and 32 mm, respectively. CONCLUSIONS: Eugenol added to cattle waste may be more beneficial than thymol because not only does it effectively control faecal coliforms and odour (VFA production), it also stimulates lactate accumulation. This in turn, causes the pH to drop more rapidly, further inhibiting microbial activity and nutrient emissions. SIGNIFICANCE AND IMPACT OF THE STUDY: Plant essential oils have the potential to solve some of the environmental problems associated with consolidated animal feeding operations. Thymol and eugenol reduce fermentative activity, thus, have the potential to reduce emissions of greenhouse gases and odour, and curtail transmission of pathogens in cattle and swine wastes.     

Commensal Interactions in a Dual-Species Biofilm Exposed to Mixed Organic Compounds

There is limited knowledge of interspecies interactions in biofilm communities. In this study, Pseudomonas sp. strain GJ1, a 2-chloroethanol (2-CE)-degrading organism, and Pseudomonas putida DMP1, a p-cresol-degrading organism, produced distinct biofilms in response to model mixed waste streams composed of 2-CE and various p-cresol concentrations. The two organisms maintained a commensal relationship, with DMP1 mitigating the inhibitory effects of p-cresol on GJ1. A triple-labeling technique compatible with confocal microscopy was used to investigate the influence of toxicant concentrations on biofilm morphology, species distribution, and exopolysaccharide production. Single-species biofilms of GJ1 shifted from loosely associated cell clusters connected by exopolysaccharide to densely packed structures as the p-cresol concentrations increased, and biofilm formation was severely inhibited at high p-cresol concentrations. In contrast, GJ1 was abundant when associated with DMP1 in a dual-species biofilm at all p-cresol concentrations, although at high p-cresol concentrations it was present only in regions of the biofilm where it was surrounded by DMP1. Evidence in support of a commensal relationship between DMP1 and GJ1 was obtained by comparing GJ1-DMP1 biofilms with dual-species biofilms containing GJ1 and Escherichia coli ATCC 33456, an adhesive strain that does not mineralize p-cresol. Additionally, the data indicated that only tower-like cell structures in the GJ1-DMP1 biofilm produced exopolysaccharide, in contrast to the uniform distribution of EPS in the single-species GJ1 biofilm.     

Effects of different mineral supplements on fertilization of phenol-contaminated soils by Corynebacterium glutamicum

This study focused on the effects of different mineral supplements on the ability of Corynebacterium glutamicum to degrade phenol in contaminated soil and convert the phenol into useful amino acids. Three types of minerals including FeSO₄, MgSO₄, and MnSO₄ were added at several concentrations to C. glutamicum culture media containing 1% yeast extract prior to treating the soil samples with 4.24 mM phenol. The reactor was incubated at 30°C and 150 rpm for 3 days, and the treated soil was sampled daily and analyzed using gas chromatography for residual phenol and the amino acids produced. Additionally, a plant toxicity assay was employed to examine the fertilization of the phenol-contaminated soil after C. glutamicum treatment supplemented with the three minerals. Our results suggested that among various tested concentrations, 72 μM of iron showed a significant effect on the utilization of phenol by C. glutamicum for conversion to amino acids, therefore enhancing fertilization of the phenol-contaminated soil.     

Production of Skatole and para-Cresol by a Rumen Lactobacillus sp.

The objective of this study was to examine the substrate specificity of several ruminal strains of a Lactobacillus sp. which previously was shown to produce skatole (3-methylindole) by the decarboxylation of indoleacetic acid. A total of 13 compounds were tested for decarboxylase activity. The Lactobacillus strains produced p-cresol (4-methylphenol) by the decarboxylation of p-hydroxyphenylacetic acid, but did not produce either o-cresol or m-cresol from the corresponding hydroxyphenylacetic acid isomers. These strains also decarboxylated 5-hydroxyindoleacetic acid to 5-hydroxyskatole and 3,4-dihydroxyphenylacetic acid to methylcatechol. Skatole and p-cresol were produced in a 0.5:1 ratio, when indoleacetic acid and p-hydroxyphenylacetic acid were combined in equimolar concentrations. Competition studies with indoleacetic acid and p-hydroxyphenylacetic acid suggested that two different decarboxylating enzymes are involved in the production of skatole and p-cresol by these strains. This is the first demonstration of both skatole production and p-cresol production by a single bacterium.     

Influence of p-cresol on the proteome of the autotrophic nitrifying bacterium Nitrosomonas eutropha C91

In this study, the effect of the organic micropollutant and known inhibitor of nitrification, p-cresol, was investigated on the metabolism of the ammonia oxidizing bacteria (AOB) Nitrosomonas eutropha C91 using MS-based quantitative proteomics. Several studies have demonstrated that AOB are capable of biotransforming a wide variety of aromatic compounds making them suitable candidates for bioremediation, yet the underlying molecular mechanisms are poorly described. The effect of two different concentrations of the aromatic micropollutant p-cresol (1 and 10 mg L^?1) on the metabolism of N. eutropha C91, relative to a p-cresol absent control, was investigated. Though the rate of nitrification in N. eutropha C91 appeared essentially unaffected at both concentrations of p-cresol relative to the control, the expressional pattern of the proteins of N. eutropha C91 changed significantly. The presence of p-cresol resulted in the repressed expression of several key proteins related to N-metabolism, seemingly impairing energy production in N. eutropha C91, contradicting the observed unaltered rates of nitrification. However, the expression of proteins of the TCA cycle and proteins related to xenobiotic degradation, including a p-cresol dehydrogenase, was found to be stimulated by the presence of p-cresol. This indicates that N. eutropha C91 is capable of degrading p-cresol and that it assimilates degradation intermediates into the TCA cycle. The results reveal a pathway for p-cresol degradation and subsequent entry point in the TCA cycle in N. eutropha C91. The obtained data indicate that mixotrophy, rather than cometabolism, is the major mechanism behind p-cresol degradation in N. eutropha C91.     

Simple high-performance liquid chromatographic analysis of phenol and p-cresol in urine and feces

Previous reports which present methods of analysis of phenol and p-cresol by HPLC are usually designed for the detection of these compounds in urine, can be complicated by the use of uncommon equipment or additional techniques such as steam distillation or derivatisation, or concentrate on the detection of phenol rather than p-cresol. In this paper we report a simple method suitable for the analysis of phenol and p-cresol in both urine and feces, based upon extraction into ether following acid hydrolysis and UV detection.     

The deleterious metabolic and genotoxic effects of the bacterial metabolite p-cresol on colonic epithelial cells

p-Cresol that is produced by the intestinal microbiota from the amino acid tyrosine is found at millimolar concentrations in the human feces. The effects of this metabolite on colonic epithelial cells were tested in this study. Using the human colonic epithelial HT-29 Glc^–/+ cell line, we found that 0.8 mM p-cresol inhibits cell proliferation, an effect concomitant with an accumulation of the cells in the S phase and with a slight increase of cell detachment without necrotic effect. At this concentration, p-cresol inhibited oxygen consumption in HT-29 Glc^–/+ cells. In rat normal colonocytes, p-cresol also inhibited respiration. Pretreatment of HT-29 Glc^–/+ cells with 0.8 mM p-cresol for 1 day resulted in an increase of the state 3 oxygen consumption and of the cell maximal respiratory capacity with concomitant increased anion superoxide production. At higher concentrations (1.6 and 3.2 mM), p-cresol showed similar effects but additionally increased after 1 day the proton leak through the inner mitochondrial membrane, decreasing the mitochondrial bioenergetic activity. At these concentrations, p-cresol was found to be genotoxic toward HT-29 Glc^–/+ and also LS-174T intestinal cells. Lastly, a decreased ATP intracellular content was observed after 3 days treatment. p-Cresol at 0.8 mM concentration inhibits colonocyte respiration and proliferation. In response, cells can mobilize their “respiratory reserve.” At higher concentrations, p-cresol pretreatment uncouples cell respiration and ATP synthesis, increases DNA damage, and finally decreases the ATP cell content. Thus, we have identified p-cresol as a metabolic troublemaker and as a genotoxic agent toward colonocytes.     

Isolation and Characterization of Thermophilic Bacilli Degrading Cinnamic, 4-Coumaric, and Ferulic Acids

Thirty-four thermophilic Bacillus sp. strains were isolated from decayed wood bark and a hot spring water sample based on their ability to degrade vanillic acid under thermophilic conditions. It was found that these bacteria were able to degrade a wide range of aromatic acids such as cinnamic, 4-coumaric, 3-phenylpropionic, 3-(p-hydroxyphenyl)propionic, ferulic, benzoic, and 4-hydroxybenzoic acids. The metabolic pathways for the degradation of these aromatic acids at 60°C were examined by using one of the isolates, strain B1. Benzoic and 4-hydroxybenzoic acids were detected as breakdown products from cinnamic and 4-coumaric acids, respectively. The β-oxidative mechanism was proposed to be responsible for these conversions. The degradation of benzoic and 4-hydroxybenzoic acids was determined to proceed through catechol and gentisic acid, respectively, for their ring fission. It is likely that a non-β-oxidative mechanism is the case in the ferulic acid catabolism, which involved 4-hydroxy-3-methoxyphenyl-β-hydroxypropionic acid, vanillin, and vanillic acid as the intermediates. Other strains examined, which are V0, D1, E1, G2, ZI3, and H4, were found to have the same pathways as those of strain B1, except that strains V0, D1, and H4 had the ability to transform 3-hydroxybenzoic acid to gentisic acid, which strain B1 could not do.     

Isolation of a microbial consortium from activated sludge for the biological treatment of food waste

The bacterial community in the activated sludge of a local wastewater treatment plant was studied in an effort to understand and exploit the metabolic versatility of microorganisms for the efficient biological treatment of food waste. Microorganisms capable of and efficient in degrading domestic food waste were screened based on their ability to produce areas of clearing on selective media containing protein, fat, cellulose and starch. Nine microbial species belonging to the genera Flavobacterium, Pseudomonas, Micrococcus, Aeromonas, Xanthomonas, Vibrio and Sphingomonas were found to degrade all components of food waste. These bacteria were added to domestic wastewater and shown to cause a 60% reduction in the biochemical oxygen demand (BOD) level of wastewater compared to a control in which no microorganisms were added. The ability of the microbial consortium to degrade domestic wastewater as evidenced by the decrease in BOD levels suggests its potential for use in the biological treatment of food waste.     

Formation of Phenolic and Indolic Compounds by Anaerobic Bacteria in the Human Large Intestine

Abstract Batch culture incubations were used to investigate the effects of pH (6.8 or 5.5) and carbohydrate (starch) availability on dissimilatory aromatic amino acid metabolism in human fecal bacteria. During growth on peptide mixtures, tyrosine and phenylalanine fermentations occurred optimally at pH 6.8, while individual metabolic reactions were inhibited by up to 80% in the presence of 10 g l⁻¹ starch. Tryptophan metabolites were not detected in these experiments. When free amino acids replaced peptides, phenol production was increased during carbohydrate fermentation, although formation of p-cresol, another tyrosine metabolite was strongly inhibited. Phenylpropionate, which is produced from phenylalanine, was unaffected by starch. Tryptophan was fermented in these studies, although indole production was reduced in the starch fermentors. The importance of different fermentation substrates (casein, peptide mixtures, free amino acids) on aromatic amino acid metabolism was investigated in incubations of material taken from the proximal bowel. The phenylalanine metabolites, phenylacetate and phenylpropionate, were the principal phenolic compounds formed from all three substrates. Phenol was the major tyrosine metabolite produced in casein and peptide fermentations, while hydroxyphenylpropionate was a more important tyrosine product from free amino acids. Indole was the sole product of tryptophan metabolism, but was formed only from the free amino acid. Bacterial metabolism of individual phenolic and indolic compounds was also investigated. Phenol, p-cresol, phenylacetate, phenylpropionate, 4-ethylphenol, indole, indoleacetate, and indolepropionate were not metabolized by colonic bacteria. However, hydroxyphenylacetate was hydrolyzed to p-cresol, while hydroxyphenylpropionate was transformed into phenylpropionate. Indolepyruvate was either converted to indoleacetate or metabolized into indole. Indolepropionate, and to a lesser degree indoleacetate were produced from indolelactate. These data show that human colonic anaerobes are able to extensively degrade either free or peptide-bound aromatic amino acids, with the concomitant formation of toxic metabolic products. These processes are controlled to a significant degree by environmental factors such as pH and carbohydrate availability, and this ultimately influences the types and amounts of fermentation products that can be formed in different regions of the large bowel. Received: 25 January 1996; Accepted: 8 May 1996     

侧耳属真菌经济利用的研究进展

侧耳属真菌分布广泛、是大型真菌中生物多样性最为丰富的类群之一。其含有丰富的多糖、蛋白质、氨基酸、脂肪酸等生物活性物质,在食品、保健和医药等方面具有广泛用途。侧耳属真菌通过产生木质素氧化酶、锰过氧化物酶和漆酶等酶类降解多环芳烃、染料、工农业废弃物等有害物,并广泛应用于纺织厂、造纸厂、橄榄油厂废水及城市废水的处理。同时,侧耳属是食用菌家族中利用基质最为广泛的真菌,可以多种农业、林业、轻工业等废弃物为基质进行栽培生产,且生物学效率较高,具重要农业生产应用价值。     

Biodegradation of phenolic compounds by sulfate-reducing bacteria from contaminated sediments

The biodegradation of phenolic compounds under sulfate-reducing conditions was studied in sediments from northern Indiana. Phenol, p-cresol and 4-chlorophenol were selected as test substrates and added to sediment suspensions from four sites at an initial concentration of 10 mg/liter. Degradative abilities of the sediment microorganisms from the four sites could be related to previous exposure to phenolic pollution. Time to onset of biodegradation of p-cresol and phenol in sediment suspensions from a nonindustrialized site was approximately 70 and 100 days, respectively, in unacclimated cultures. In sediment slurries from three sites with a history of wastewater discharges containing phenolics, time to onset of biodegradation was 50–70 days for p-cresol and 50–70 days for phenol in unacclimated cultures. In acclimated cultures from all four sites, the length of the lag phase was reduced to 14–35 days for p-cresol and 25–60 days for phenol. Length of the biodegradative phase varied from 25 to 40 days for phenol and 10 to 50 days for p-cresol and was not markedly affected by acclimation. Substrate mineralization by sulfate-reducing bacteria was confirmed with radiotracer techniques using an acclimated sediment culture from one site. Addition of molybdate, a specific inhibitor of sulfate reduction, and bacterial cell inactivation inhibited sulfate reduction and substrate utilization. None of the sites exhibited the ability to degrade 4-chlorophenol, nor were acclimated phenol and p-cresol degrading cultures from a particular site able to cometabolize 4-chlorophenol.Correspondence to: D. Dean-Ross     

Construction of a Corynebacterium glutamicum platform strain for the production of stilbenes and (2S)-flavanones

Corynebacterium glutamicum is an important organism in industrial biotechnology for the microbial production of bulk chemicals, in particular amino acids. However, until now activity of a complex catabolic network for the degradation of aromatic compounds averted application of C. glutamicum as production host for aromatic compounds of pharmaceutical or biotechnological interest. In the course of the construction of a suitable C. glutamicum platform strain for plant polyphenol production, four gene clusters comprising 21 genes involved in the catabolism of aromatic compounds were deleted. Expression of plant-derived and codon-optimized genes coding for a chalcone synthase (CHS) and a chalcone isomerase (CHI) in this strain background enabled formation of 35 mg/L naringenin and 37 mg/L eriodictyol from the supplemented phenylpropanoids p-coumaric acid and caffeic acid, respectively. Furthermore, expression of genes coding for a 4-coumarate: CoA-ligase (4CL) and a stilbene synthase (STS) led to the production of the stilbenes pinosylvin, resveratrol and piceatannol starting from supplemented phenylpropanoids cinnamic acid, p-coumaric acid and caffeic acid, respectively. Stilbene concentrations of up to 158 mg/L could be achieved. Additional engineering of the amino acid metabolism for an optimal connection to the synthetic plant polyphenol pathways enabled resveratrol production directly from glucose. The construction of these C. glutamicum platform strains for the synthesis of plant polyphenols opens the door towards the microbial production of high-value aromatic compounds from cheap carbon sources with this microorganism.     

Initiation of anaerobic degradation of p-cresol by formation of 4-hydroxybenzylsuccinate in desulfobacterium cetonicum

The anaerobic bacterium Desulfobacterium cetonicum oxidized p-cresol completely to CO₂ with sulfate as the electron acceptor. During growth, 4-hydroxybenzylsuccinate accumulated in the medium. This finding indicated that the methyl group of p-cresol is activated by addition to fumarate, analogous to anaerobic toluene, m-xylene, and m-cresol degradation. In cell extracts, the formation of 4-hydroxybenzylsuccinate from p-cresol and fumarate was detected at an initial rate of 0.57 nmol min⁻¹ (mg of protein)⁻¹. This activity was specific for extracts of p-cresol-grown cells. 4-Hydroxybenzylsuccinate was degraded further to 4-hydroxybenzoyl-coenzyme A (CoA), most likely via β-oxidation. 4-Hydroxybenzoyl-CoA was reductively dehydroxylated to benzoyl-CoA. There was no evidence of degradation of p-cresol via methyl group oxidation by p-cresol-methylhydroxylase in this bacterium.     

Biological sludge reduction and enhanced nutrient removal in a pilot-scale system with 2-step sludge alkaline fermentation and A2O process

To enhance nutrient removal performance and reduce disposal amount of waste activated sludge (WAS), a pilot-scale continuous system consisting of a 2-step sludge alkaline fermentation process and an A²O reactor was proposed. The feasibility of WAS reducing and resourcing by alkaline fermentation was investigated. Volatile fatty acids (VFA) yield was higher under alkaline condition than that under acidic condition. Through 2-step alkaline fermentation, substantial VFA was accumulated, and then elutriated out continuously from an up-flow column by domestic wastewater. The results showed that 38.2% of sludge was hydrolyzed, 19.7% was finally acidified into VFA, and as high as 42.1% of WAS was reduced. Moreover, after introducing the fermentation liquids with higher proportion of acetic acid and propionic acid into the A²O reactor, the total nitrogen and phosphorus removal efficiencies reached to 80.1% and 90.0%, respectively. Sludge reduction and enhanced nutrient removal could be achieved simultaneously in the proposed system.