The role of the combined nitrogen-sulfur-carbon cycles for efficient performance of anammox-based systems.

The combined anammox/mixotrophic denitrification process was conducted in two granular sequencing batch reactors (SBRs) during a 200-day operation. Both reactors were fed with synthetic medium, but SBR2 was enriched with additional sulfate (SO42-) which influenced sulfate reduction ammonium oxidation (SRAO) and heterotrophic reduction of SO42- by sulfate reducing bacteria. It was hypothesized that the addition of SO42- could positively impact the removal rates of N-S-C compounds. A low C/N ratio (0.4-1.6) was maintained to prevent inhibition of anaerobic ammonium oxidizing bacteria (AnAOB), and alternating chemical oxygen demand (COD) on/off conditions were used to regenerate AnAOB during COD-off phases and heterotrophic denitrifiers during COD-on phases. Stoichiometric analysis showed that introducing SO42- in SBR2 enhanced the ammonium utilization rate, which was approximately 10 % higher compared to SBR1 in the final stage of the experiment (25.8 vs. 22.8 mg N/(g VSS·h)). The total nitrogen removal efficiencies ranged from 62 % to 99 % in both reactors, with SBR2 consistently exhibiting approximately 4 % higher efficiency than SBR1. In SBR2, the maximum overall SO42- utilization efficiency reached 27 % under COD-off conditions, while overall COD utilization was almost complete under COD-on conditions. A strong correlation (R2 = 0.98) was observed between SO42- production and COD utilization. The key players responsible for N and S transformations in response to SO42- addition were Candidatus Brocadia and Chloroflexi - Anaerolineae. This study highlights the potential to enhance the overall efficiency of N-S-C removal by implementing an integrated anammox/mixotrophic denitrification process. The combination of cycles emerges as a sustainable approach for treating wastewater rich in N-S-C compounds.

Application of PAT Probes in Aluminum Phosphate Adjuvant Manufacturing.

PURPOSE: This study is focused on monitoring process parameters and quality attributes of aluminum phosphate (AlPO4) using multiple in-line probes incorporated into an industrial-scale adjuvant suspension manufacturing unit. METHODS: The manufacturing of aluminum adjuvant suspension was monitored at manufacturing scale using conductivity, turbidity, infrared, and particle sizing and count probes to follow the continuous evolution of particle formation and size distribution, and the reaction kinetics during the synthesis of AlPO4. RESULTS: The data showed that AlPO4 forms large particles at the early stages of mixing, followed by a decrease in size and then stabilization towards the later stages of mixing and pH adjustment. The results provided a complementary view of process events and assisted in optimizing several parameters, e.g., flow rate of reactants AlCl3 and Na3PO4 solutions, mixing rate, pH, and conductivity of AlPO4, as well as adjuvant quality attribute such as particle size, thus streamlining and shortening the process development stage. CONCLUSION: The results of this study showed the usefulness of the in-line probes to automate continuous assessment of AlPO4 batch-to-batch consistency during in-house adjuvant production at the industrial scale.

Integration of the sulfate reduction and anammox processes for enhancing sustainable nitrogen removal in granular sludge reactors.

The Anammox and Sulfate Reduction Ammonium Oxidation processes were compared in two granular sequencing batch reactors operated for 160 days under anammox conditions. It was hypothesized that increasing the concentration of SO42- may positively influence the rate of N removal under anaerobic conditions and it was tested whether SO42- reduction and anammox occur independently or are related to each other. The cooperation of N-S cycles by increasing the concentration of influent SO42- to 952 mg S/L in the second reactor, a higher ammonium utilization rate and sulfate utilization rate was achieved compared to the first reactor, i.e., 2.1-fold and 15-fold, respectively. Nitrosomonas played the dominant role in the N metabolism, while Thauera - in the S metabolism. This study highlights the benefits of linking the N-S cycles as an effective approach for the treatment of NH4+ and SO42- - rich wastewater, including lower substrate removal cost and reduced energy consumption.

The coexistence and competition of canonical and comammox nitrite oxidizing bacteria in a nitrifying activated sludge system - Experimental observations and simulation studies.

The second step of nitrification can be mediated by nitrite oxidizing bacteria (NOB), i.e. Nitrospira and Nitrobacter, with different characteristics in terms of the r/K theory. In this study, an activated sludge model was developed to account for competition between two groups of canonical NOB and comammox bacteria. Heterotrophic denitrification on soluble microbial products was also incorporated into the model. Four 5-week washout trials were carried out at dissolved oxygen-limited conditions for different temperatures (12 °C vs. 20 °C) and main substrates (NH4+-N vs. NO2--N). Due to the aggressive reduction of solids retention time (from 4 to 1 d), the biomass concentrations were continuously decreased and stabilized after two weeks at a level below 400 mg/L. The collected experimental data (N species, biomass concentrations, and microbiological analyses) were used for model calibration and validation. In addition to the standard predictions (N species and biomass), the newly developed model also accurately predicted two microbiological indicators, including the relative abundance of comammox bacteria as well as nitrifiers to heterotrophs ratio. Sankey diagrams revealed that the relative contributions of specific microbial groups to N conversion pathways were significantly shifted during the trial. The contribution of comammox did not exceed 5 % in the experiments with both NH4+-N and NO2--N substrates. This study contributes to a better understanding of the novel autotrophic N removal processes (e.g. deammonification) with nitrite as a central intermediate product.

New insights into modeling two-step nitrification in activated sludge systems - The effects of initial biomass concentrations, comammox and heterotrophic activities.

In this study, the conventional two-step nitrification model was extended with complete ammonia oxidation (comammox) and heterotrophic denitrification on soluble microbial products. The data for model calibration/validation were collected at four long-term washout experiments when the solid retention time (SRT) and hydraulic retention time (HRT) were progressively reduced from 4 d to 1 d, with mixed liquor suspended solids (MLSS) of approximately 2000 mg/L at the start of each trial. A new calibration protocol was proposed by including a systematic calculation of the initial biomass concentrations and microbial relationships as the calibration targets. Moreover, the impact assessment of initial biomass concentrations (X) and maximum growth rates (µ) for ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), comammox Nitrospira, and heterotrophs on the calibration accuracy were investigated using the response surface methodology (RSM). The RSM results revealed the strongest interaction of XAOB and µAOB on the model calibration accuracy. All the examined model efficiency measures confirmed that the extended model was accurately calibrated and validated. The estimated µ values were as follows: µAOB = 0.38 ± 0.005 d-1, µNOB = 0.20 ± 0.01 d-1, µCMX = 0.20 ± 0.01 d-1, µHET = 1.0 ± 0.03 d-1. For comparison, when using the conventional model, µAOB and µNOB increased respectively by 26 and 15 % (µAOB = 0.48 ± 0.02 d-1 and µNOB = 0.23 ± 0.005 d-1). This study provides better understanding of the effects of the initial biomass composition and the accompanying processes (comammox and heterotrophic denitrification) on modeling two-step nitrification.

Rearrangements of the nitrifiers population in an activated sludge system under decreasing solids retention times.

Due to the key role of nitrite in novel nitrogen removal systems, nitrite oxidizing bacteria (NOB) have been receiving increasing attention. In this study, the coexistence and interactions of nitrifying bacteria were explored at decreasing solids retention times (SRTs). Four 5-week washout experiments were carried out in laboratory-scale (V = 10 L) sequencing batch reactors (SBRs) with mixed liquor from two full-scale activated sludge systems (continuous flow vs SBR). During the experiments, the SRT was gradually reduced from the initial value of 4.0 d to approximately 1.0 d. The reactors were operated under limited dissolved oxygen conditions (set point of 0.6 mg O2/L) and two process temperatures: 12 °C (winter) and 20 °C (summer). At both temperatures, the progressive SRT reduction was inefficient for the out-selection of both canonical NOB and comammox Nitrospira. However, the dominant NOB switched from Nitrospira to Ca. Nitrotoga, whereas the dominant AOB was always Nitrosomonas. The results of this study are important for optimizing NOB suppression strategies in the novel N removal processes, which are based on nitrite accumulation.

Monitoring Lead Concentration in the Surrounding Environmental Components of a Lead Battery Company: Plants, Air and Effluents-Case Study, Kenya.

Lead (Pb) pollution from smelters and lead-acid battery has become a serious problem worldwide owing to its toxic nature as a heavy metal. Stricter regulations and monitoring strategies have been formulated, legislated and implemented in various parts of the world on heavy metal usage. Developed countries such as the USA and in Europe largely operate within the set standards, however, developing countries such as Kenya, Nigeria and India, with limited regulatory capacity, resources and sufficient data, face poor Pb waste management and exposure of the population to health risks. This study assessed the pollution concerns from Associated Battery Manufacturers (East Africa) Limited (ABM), located in the Nairobi Industrial Area in Kenya. Samples of air, extracts from plants (leaves) and factory wastewaters were taken from different operations units, prepared and analysed with Atomic Absorption Spectrometry (AAS). Pb traces remained fairly controlled with averages of 1.24 ± 0.42 parts per million (ppm), 1.21 ± 0.02 ppm and 0.29 ± 0.01 ppm in the air, plant extracts and effluents, respectively. The conducted research shows that the obtained lead concentrations in the air, wastewater and surrounding plants exceeded the recommended standards, and are potentially harmful not only to workers, but also to the surrounding villages.

Incorporation of the complete ammonia oxidation (comammox) process for modeling nitrification in suspended growth wastewater treatment systems.

The newly discovered process complete ammonia oxidation (comammox) has changed the traditional understanding of nitrification. In this study, three possible concepts of comammox were developed and incorporated as part of an extended two-step nitrification model. For model calibration and validation, two series of long-term biomass washout experiments were carried out at 12 °C and 20 °C in a laboratory sequencing batch reactor. The inoculum biomass was withdrawn from a large biological nutrient removal wastewater treatment plant. The efficiency of the examined models was compared based on the behaviors of ammonia, nitrite, and nitrate in the studied reactor. Predictions of the conventional approach to comammox, assuming the direct oxidation of ammonia to nitrate, were slightly better than the two other approaches. Simulation results revealed that comammox could be responsible for the conversion of >20% of the influent ammonia load. Therefore, the role of commamox in the nitrogen mass balance in activated sludge systems should not be neglected and requires further investigation. Furthermore, sensitivity and correlation analysis revealed that the maximum growth rates (µ), oxygen half-saturation (KO), and decay rates (b) of the canonical nitrifiers and comammox were the most sensitive factors, and the highest correlation was found between µ and b among all considered kinetic parameters. The estimated µ values by the best model were 0.57, 0.11, and 0.15 d-1 for AOB, NOB, and comammox bacteria, respectively.

Optimization of the Aeration Strategies in a Deammonification Sequencing Batch Reactor for Efficient Nitrogen Removal and Mitigation of N2O Production.

In deammonification systems, nitrite-oxidizing bacteria (NOB) suppression and nitrous oxide (N2O) mitigation are two important operational objectives. To carry out this multivariable analysis of response, a comprehensive model for the N cycle was developed and evaluated against experimental data from a laboratory-scale deammonification granular sludge sequencing batch reactor. Different aeration strategies were tested, and the manipulated variables comprised the dissolved oxygen (DO) set point in the aerated phase, aeration on/off frequency (F), and the ratio (R) between the non-aerated and aerated phase durations. Experimental results showed that a high ammonium utilization rate (AUR) in relation to the low nitrate production rate (NPR) (NPR/AUR = 0.07-0.08) and limited N2O emissions (EN2O < 2%) could be achieved at the DO set point = 0.7 mg O2/L, R ratio = 2, and F frequency = 6-7 h-1. Under specific operational conditions (biomass concentration, NH4+-N loading rate, and temperature), simulation results confirmed the feasible aeration strategies for the trade-offs between the NOB suppression and N2O emission. The intermittent aeration regimes led to frequent shifts in the predominating N2O production pathways, that is, hydroxylamine (NH2OH) oxidation (aerated phase) versus autotrophic denitrification (non-aerated phase). The inclusion of the extracellular polymeric substance mechanism in the model explained the observed activity of heterotrophs, especially Anaerolineae, and granule formation.

Assessment of diversity and composition of bacterial community in sludge treatment reed bed systems.

Due to their low emission of odours and lack of the need to apply additional chemical agents, sludge treatment reed beds (STRBs) constitute an economically feasible and eco-friendly approach to sewage sludge management. Correctly designed and operated STRBs ensure effective reduction of the dry matter content coupled with the mineralisation of organic compounds. Successful operation of STRBs relies on complex interactions between the plants and microorganisms responsible for the decomposition of organic matter and nutrient cycling. While the biocenoses of wetland systems dedicated to wastewater treatment have been intensively investigated, in the case of sludge treatment applications, there is a deficit of available microbial data. The aim of this study was to explore the diversity and spatial distribution of the bacteria in three distinct STRBs which differ in maturation and feeding patterns. Analyses of the dry mass and organic matter content showed the general trend of the sludge stabilisation processes advancing through the bed depth, with the best performance in the Matured Continuous Feed (MCF) bed being noted. Samples from the MCF bed showed the statistically greatest biodiversity in relation to the other beds. Moreover, increased biodiversity of microorganisms was observed on the surface of the STRBs and the bottom zone of the MCF equipped with a passive aeration system, which proves the application of such solutions in order to enhance the performance of the process. The results of 16S rRNA gene sequencing revealed that Bacteroidetes, Proteobacteria and Firmicutes contributed approximately 80% of all identified sequences read. Network analysis revealed dominant role of Bacteroidetes in the formation of interspecies co-existence patterns. Nitrospira was the most abundant organism responsible for nitrogen metabolism in the STRBs.

The occurrence and role of Nitrospira in nitrogen removal systems.

Application of the modern microbial techniques changed the paradigm about the microorganisms performing nitrification. Numerous investigations recognized representatives of the genus Nitrospira as a key and predominant nitrite-oxidizing bacteria in biological nutrient removal systems, especially under low dissolved oxygen and substrate conditions. The recent discovery of Nitrospira capable of performing complete ammonia oxidation (comammox) raised a fundamental question about the actual role of Nitrospira in both nitrification steps. This review summarizes the current knowledge about morphological, physiological and genetic characteristics of the canonical and comammox Nitrospira. Potential implications of comammox for the functional aspects of nitrogen removal have been highlighted. The complex meta-analysis of literature data was applied to identify specific individual variables and their combined interactions on the Nitrospira abundance. In addition to dissolved oxygen and influent nitrogen concentrations, temperature and pH may play an important role in enhancing or suppressing the Nitrospira activity.

The metabolic activity of denitrifying microorganisms accumulating polyphosphate in response to addition of fusel oil.

The effect of distillery waste product (fusel oil) as an alternative external organic carbon source (EOCS) was investigated in terms of the metabolic properties of denitrifying polyphosphate accumulating organisms (DPAOs). Samples of the non-acclimated biomass were collected from a local full-scale wastewater treatment plant employing A2/O type bioreactors. The acclimated biomass was obtained after cultivation (with fusel oil added) in a bench-scale reactor with a process configuration similar to the full-scale bioreactor. Changes in the functional properties of the biomass were investigated by measuring the phosphate release/uptake rates (PRRs and PURs), and nitrate utilization rates (NURs) with fusel oil in anaerobic-anoxic batch tests. Furthermore, a validated extended Activated Sludge Model no 2d (ASM2d) was used as a supporting tool to analyze the experimental results and estimate the contribution of DPAOs to the overall denitrification. In the non-acclimated biomass with fusel oil, the PRRs, PURs and NURs were low and close to the rates obtained in a reference test without adding EOCS. With the acclimated biomass, the PUR and NUR increased significantly, i.e., 3.5 and 2.7 times, respectively. In the non-acclimated biomass, approximately 60.0 ± 3.6% and 20.0 ± 2.2% of the total NUR was attributed to the utilization of endogenous carbon and examined EOCS, respectively. The remaining portion (20% of the total NUR) was attributed to PHA utilization (linked to PO4-P uptake) by DPAOs. With the acclimated biomass, the contribution of the EOCS to the NUR increased to approximately 60%, while the contribution of the endogenous carbon source decreased accordingly. Very accurate predictions of PURs and NURs (R2 = 0.97-1.00) were obtained with the extended ASM2d. Based on model simulations, it was estimated that the activity of DPAOs and denitrifying ordinary heterotrophic organisms corresponded to approximately 20% and 80% of the total NUR, respectively.

Acclimation of denitrifying activated sludge to a single vs. complex external carbon source during a start-up of sequencing batch reactors treating ammonium-rich anaerobic sludge digester liquors.

In this study, denitrification of ammonium-reach anaerobic sludge digester liquor was investigated during start-up periods of two laboratory-scale "fill-and-draw" reactors. One reactor was fed with a single carbon source (ethanol), whereas the other reactor was fed with a complex carbon source (fusel oil). During two acclimation experiments, the structure of microbial community involved in denitrification was analyzed using 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis fingerprints and fluorescent in situ hybridization. The characteristics of the mixed liquor were additionally supported by regular measurements of nitrate uptake rates. The addition of fusel oil and ethanol resulted in a significant enhancement of the denitrification rate and efficiency combined with the increasing volumetric addition of sludge digester liquor up to 15 % of the reactor volume. The microbiological analyses revealed that the addition of sludge digester liquor as well as both external carbon sources (fusel oil and ethanol) did not affect the structure of microbial communities in a severe way. In both reactors, Curvibacter sp. and Azoarcus sp. were found as the most abundant representatives of denitrifiers.

Ribosomal intergenic spacer analysis as a tool for monitoring methanogenic Archaea changes in an anaerobic digester.

The applicability of a newly-designed PCR primer pair in examination of methanogenic Archaea in a digester treating plant biomass was evaluated by Ribosmal Intergenic Spacer Analysis (RISA). To find a suitable approach, three variants of RISA were tested: (1) standard, polyacrylamide gel-based, (2) automated, utilized capillary electrophoresis (GA-ARISA), and (3) automated microfluidics-based (MF-ARISA). All three techniques yielded a consistent picture of archaeal community structure changes during anaerobic digestion monitored for more than 6 weeks. While automated variants were more practical for handling and rapid analysis of methanogenic Archaea, the gel-based technique was advantageous when micro-organism identification was required. A DNA-sequence analysis of dominant bands extracted from the gel revealed that the main role in methane synthesis was played by micro-organisms affiliated with Methanosarcina barkeri. The obtained results revealed that RISA is a robust method allowing for detailed analysis of archaeal community structure during organic biomass conversion into biogas. In addition, our results showed that GA-ARISA has a higher resolution and reproducibility than other variants of RISA and could be used as a technique for tracking changes in methanogenic Archaea in an anaerobic digester.

Characterization of bacterial structures in two-stage moving-bed biofilm reactor (MBBR) during nitrification of the landfill leachate.

Differences in DNA banding patterns, obtained by ribosomal intergenic spacer analysis (RISA), and nitrification were followed in a moving-bed biofilm reactor (MBBR) receiving municipal landfill leachate. Complete nitrification (> 99%) to nitrate was obtained in the two-stage MBBR system with an ammonium load of 1.09 g N-NH(4)/m(2).d. Increasing the ammonium load to 2.03 g N-NH(4)/m(2).d or more caused a decline in process efficiency to 70-86%. Moreover, at the highest ammonium load (3.76 g N-NH(4)/m(2).d), nitrite was the predominant product of nitrification. Community succession was evident in both compartments in response to changes in ammonium load. Non-metric multidimensional scaling (NMDS) supported by similarity analysis (ANOSIM) showed that microbial biofilm communities differed between compartments. The microbial biofilm was composed mainly of ammonia-oxidizing bacteria (AOB), with Nitrosomonas europeae and N. eutropha being most abundant. These results suggest that high ammonium concentrations select for particular AOB strains.

Municipal landfill leachate nitrification in RBC biofilm - process efficiency and molecular analysis of microbial structure.

Landfill leachates were treated using a two-stage rotating biological contactor (RBC). At an ammonium load of 1.92gN-NH(4)/m(2)d, complete nitrification was obtained at the first stage; however, at a higher load (3.6gN-NH(4)/m(2)d) a two-stage system was needed to obtain complete nitrification. A further increase in the ammonium load to 4.79gN-NH(4)/m(2)d and 6.63gN-NH(4)/m(2)d caused a decrease of overall nitrification efficiency to 74.4% and 71.6%, respectively. Randomly amplified DNA analysis of bacteria present in the RBC revealed that the bacterial community differed over time. Regardless of the ammonium load, Nitrosomonas europaea and Nitrosomonas eutropha were the dominating species. The performed analyses provide a clear picture of bacterial population changes in response to changes of ammonium load during landfill leachate treatment.

A UAF1-containing multisubunit protein complex regulates the Fanconi anemia pathway.

The deubiquitinating enzyme USP1 controls the cellular levels of the DNA damage response protein Ub-FANCD2, a key protein of the Fanconi anemia DNA repair pathway. Here we report the purification of a USP1 multisubunit protein complex from HeLa cells containing stoichiometric amounts of a WD40 repeat-containing protein, USP1 associated factor 1 (UAF1). In vitro reconstitution of USP1 deubiquitinating enzyme activity, using either ubiquitin-7-amido-4-methylcoumarin (Ub-AMC) or purified monoubiquitinated FANCD2 protein as substrates, demonstrates that UAF1 functions as an activator of USP1. UAF1 binding increases the catalytic turnover (kcat) but does not increase the affinity of the USP1 enzyme for the substrate (KM). Moreover, we show that DNA damage results in an immediate shutoff of transcription of the USP1 gene, leading to a rapid decline in the USP1/UAF1 protein complex. Taken together, our results describe a mechanism of regulation of the deubiquitinating enzyme, USP1, and of DNA repair.

Structural determinants of human FANCF protein that function in the assembly of a DNA damage signaling complex.

Fanconi anemia (FA) is a rare autosomal recessive and X-linked chromosomal instability disorder. At least eight FA proteins (FANCA, B, C, E, F, G, L, and M) form a nuclear core complex required for monoubiquitination of a downstream protein, FANCD2. The human FANCF protein reportedly functions as a molecular adaptor within the FA nuclear complex, bridging between the subcomplexes A:G and C:E. Our x-ray crystallographic studies of the C-terminal domain of FANCF reveal a helical repeat structure similar to the Cand1 regulator of the Cul1-Rbx1-Skp1-Fbox(Skp2) ubiquitin ligase complex. Two C-terminal loops of FANCF are essential for monoubiquitination of FANCD2 and normal cellular resistance to the DNA cross-linking agent mitomycin C. FANCF mutants bearing amino acid substitutions in this C-terminal surface fail to interact with other components of the FA complex, indicating that this surface is critical for the proper assembly of the FA core complex.

Alpha-Galactosyl trisaccharide epitope: Modification of the 6-primary positions and recognition by human anti-alphaGal antibody.

Galactose oxidase (EC 1.1.3.9, GAO) was used to convert the C-6' OH of Galbeta(1 --> 4)Glcbeta-OBn (5) to the corresponding hydrated aldehyde (7). Chemical modification, through dehydratative coupling and reductive amination, gave rise to a small library of Galbeta(1 --> 4)Glcbeta-OBn analogues (9a-f, 10, 11). UDP-[6-(3)H]Gal studies indicated that alpha1,3-galactosyltransferase recognized the C-6' modified Galbeta(1 --> 4)Glcbeta-OBn analogues (9a-f, 10, 11). Preparative scale reactions ensued, utilizing a single enzyme UDP-Gal conversion as well as a dual enzymatic system (GalE and alpha1,3GalT), taking full advantage of the more economical UDP-Glc, giving rise to compounds 6, 15-22. Galalpha(1 --> 3)Galbeta(1 --> 4)Glcbeta-OBn trisaccharide (6) was produced on a large scale (2 g) and subjected to the same chemoenzymatic modification as stated above to produce C-6" modified derivatives (23-30). An ELISA bioassay was performed utilizing human anti-alphaGal antibodies to study the binding affinity of the derivatized epitopes (6, 15-30). Modifications made at the C-6' position did not alter the IgG antibody's ability to recognize the unnatural epitopes. Modifications made at the C-6" position resulted in significant or complete abrogation of recognition. The results indicate that the C-6' OH of the alphaGal trisaccharide epitope is not mandatory for antibody recognition.

Large-scale synthesis of globotriose derivatives through recombinant E. coli.

The carbohydrate chains decorating cell membranes and secreted proteins participate in a range of important biological processes. However, their ultimate significance and possible therapeutic potential have not been fully explored due to the lack of economical methods for their production. This study is an example of the use of a genetically engineered bacterial strain in the preparation of diverse oligosaccharides. Based on an ex vivo biosynthetic pathway, an artificial gene cluster was constructed by linking the genes of five associated enzymes on a plasmid vector. This plasmid was inserted into the E. coli NM522 strain to form globotriose-producing cells ('superbug' pLDR20-CKTUF). The specific strain was conveniently applied to the synthesis of globotriose trisaccharide and its derivatives, as potential neutralizers for Shiga toxin. This work demonstrates a novel and economical method for generating ligand diversity for carbohydrate drug development.