Structural insight into the subclass B1 metallo-ß-lactamase AFM-1.

The emergence of drug-resistant bacteria, facilitated by metallo-beta-lactamases (MBLs), presents a significant obstacle to the effective use of antibiotics in the management of clinical drug-resistant bacterial infections. AFM-1 is a MBL derived from Alcaligenes faecalis and shares 86% homology with the NDM-1 family. Both AFM-1 and NDM-1 demonstrate the ability to hydrolyze ampicillin and other ß-lactam antibiotics, however, their substrate affinities vary, and the specific reason for this variation remains unknown. We present the high-resolution structure of AFM-1. The active center of AFM-1 binds two zinc ions, and the conformation of the key amino acid residues in the active center is in accordance with that of NDM-1. However, the substrate-binding pocket of AFM-1 is considerably smaller than that of NDM-1. Additionally, the mutation of amino acid residues in the Loop3 region, as compared to NDM-1, results in the formation of a dense hydrophobic patch comprised of hydrophobic amino acid residues in this area, which facilitates substrate binding. Our findings lay the foundation for understanding the molecular mechanism of AFM-1 with a high affinity for substrates and provide a novel theoretical foundation for addressing the issue of drug resistance caused by B1 MBLs.

Enhanced wheat productivity in saline soil through the combined application of poultry manure and beneficial microbes.

BACKGROUND: Soil salinity is one of the major menaces to food security, particularly in dealing with the food demand of the ever-increasing global population. Production of cereal crops such as wheat is severely affected by soil salinity and improper fertilization. The present study aimed to examine the effect of selected microbes and poultry manure (PM) on seedling emergence, physiology, nutrient uptake, and growth of wheat in saline soil. A pot experiment was carried out in research area of Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan. Saline soil (12 dS m- 1 w/w) was developed by spiking using sodium chloride, and used in experiment along with two microbial strains (i.e., Alcaligenes faecalis MH-2 and Achromobacter denitrificans MH-6) and PM. Finally, wheat seeds (variety Akbar-2019) were sown in amended and unamended soil, and pots were placed following a completely randomized design. The wheat crop was harvested after 140 days of sowing. RESULTS: The results showed a 10-39% increase (compared to non-saline control) in agronomic, physiological, and nutritive attributes of wheat plants when augmented with PM and microbes. Microbes together with PM significantly enhanced seedling emergence (up to 38%), agronomic (up to 36%), and physiological (up to 33%) in saline soil as compared to their respective unamended control. Moreover, the co-use of microbes and PM also improved soil's physicochemical attributes and enhanced N (i.e., 21.7%-17.1%), P (i.e., 24.1-29.3%), and K (i.e., 28.7%-25.3%) availability to the plant (roots and shoots, respectively). Similarly, the co-use of amendments also lowered the Na+ contents in soil (i.e., up to 62%) as compared to unamended saline control. This is the first study reporting the effects of the co-addition of newly identified salt-tolerant bacterial strains and PM on seedling emergence, physiology, nutrient uptake, and growth of wheat in highly saline soil. CONCLUSION: Our findings suggest that co-using a multi-trait bacterial culture and PM could be an appropriate option for sustainable crop production in salt-affected soil.

Nanosponge hydrogel of octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate of Alcaligenes faecalis.

Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate (ODHP) was extracted in a previous study from the culture broth of soil isolate Alcaligenes faecalis MT332429 and showed a promising antimycotic activity. This study was aimed to formulate ODHP loaded ß-cyclodextrins (CD) nanosponge (NS) hydrogel (HG) to control skin fungal ailments since nanosponges augment the retention of tested agents in the skin. Box-Behnken design was used to produce the optimized NS formulation, where entrapment efficiency percent (EE%), polydispersity index (PDI), and particle size (PS) were assigned as dependent parameters, while the independent process parameters were polyvinyl alcohol % (w/v %), polymer-linker ratio, homogenization time, and speed. The carbopol 940 hydrogel was then created by incorporating the nanosponges. The hydrogel fit Higuchi's kinetic release model the best, according to in vitro drug release. Stability and photodegradation studies revealed that the NS-HG remained stable under tested conditions. The formulation also showed higher in vitro antifungal activity against Candida albicans compared to the control fluconazole. In vivo study showed that ODHP-NS-HG increased survival rates, wound contraction, and healing of wound gap and inhibited the inflammation process compared to the other control groups. The histopathological examinations and Masson's trichrome staining showed improved healing and higher records of collagen deposition. Moreover, the permeability of ODHP-NS-HG was higher through rats' skin by 1.5-folds compared to the control isoconazole 1%. Therefore, based on these results, NS-HG formulation is a potential carrier for enhanced and improved topical delivery of ODHP. Our study is a pioneering research on the development of a formulation for ODHP produced naturally from soil bacteria. KEY POINTS: • Octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propanoate was successfully formulated as a nanosponge hydrogel and statistically optimized. • The new formula exhibited in vitro good stability, drug release, and higher antifungal activity against C. albicans as compared to the fluconazole. • Ex vivo showed enhanced skin permeability, and in vivo analysis showed high antifungal activity as evidenced by measurement of various biochemical parameters and histopathological examination.

In vitro and in vivo screening of bacterial species from contaminated soil for heavy metal biotransformation activity.

Heavy metals (HMs) are widely used in various industries. High concentrations of HMs can be severely toxic to plants, animals and humans. Microorganism-based bioremediation has shown significant potential in degrading and detoxifying specific HM contaminants. In this study, we cultivated a range of bacterial strains in liquid and solid nutrient medium containing different concentrations of different HMs to select and analyze bacteria capable of transforming HMs. The bacterial strains most resistant to selected HMs and exhibiting the ability to remove HMs from contaminated soils were identified. Then, the bacterial species capable of utilizing HMs in soil model experiments were selected, and their ability to transform HMs was evaluated. This study has also generated preliminary findings on the use of plants for further removal of HMs from soil after microbial bioremediation. Alcaligenes faecalis, Delftia tsuruhatensis and Stenotrophomonas sp. were selected for their ability to grow in and utilize HM ions at the maximum permissible concentration (MPC) and two times the MPC. Lysinibacillus fusiformis (local microflora) can be used as a universal biotransformation tool for many HM ions. Brevibacillus parabrevis has potential for the removal of lead ions, and Brevibacillus reuszeri and Bacillus safensis have potential for the removal of arsenic ions from the environment. The bacterial species have been selected for bioremediation to remove heavy metal ions from the environment.

The MocR family transcriptional regulator DnfR has multiple binding sites and regulates Dirammox gene transcription in Alcaligenes faecalis JQ135.

Microbial ammonia oxidation is vital to the nitrogen cycle. A biological process, called Dirammox (direct ammonia oxidation, NH3 →NH2 OH→N2 ), has been recently identified in Alcaligenes ammonioxydans and Alcaligenes faecalis. However, its transcriptional regulatory mechanism has not yet been fully elucidated. The present study characterized a new MocR-like transcription factor DnfR that is involved in the Dirammox process in A. faecalis strain JQ135. The entire dnf cluster was composed of 10 genes and transcribed as five transcriptional units, that is, dnfIH, dnfR, dnfG, dnfABCDE and dnfF. DnfR activates the transcription of dnfIH, dnfG and dnfABCDE genes, and represses its own transcription. The intact 1506-bp dnfR gene was required for activation of Dirammox. Electrophoretic mobility shift assays and DNase I footprinting analyses showed that DnfR has one binding site in the dnfH-dnfR intergenic region and two binding sites in the dnfG-dnfA intergenic region. Three binding sites of DnfR shared a 6-bp repeated conserved sequence 5'-GGTCTG-N17 -GGTCTG-3' which was essential for the transcription of downstream target genes. Cysteine and glutamate act as possible effectors of DnfR to activate the transcription of transcriptional units of dnfG and dnfABCDE, respectively. This study provided new insights in the transcriptional regulation mechanism of Dirammox by DnfR in A. faecalis JQ135.

Optimization of a medium composition for the heterologous production of Alcaligenes faecalis penicillin G acylase in Bacillus megaterium.

Penicillin G acylase (PGA) is a strategic enzyme in the production processes of beta-lactam antibiotics. High demand for ß-lactam semisynthetic antibiotics explain the genetic and biochemical engineering strategies devoted towards novel ways for PGA production and application. This work presents a fermentation process for the heterologous production of PGA from Alcaligenes faecalis in Bacillus megaterium with optimization. The thermal stability from A. faecalis PGA is considerably higher than other described PGA and the recombinant enzyme is secreted to the culture medium by B. megaterium, which facilitates the separation and purification steps. Media optimization using fractional factorial design experiments was used to identify factors related to PGA activity detection in supernatant and cell lysates. The optimized medium resulted in almost 6-fold increased activity in the supernatant samples when compared with the basal medium. Maximum enzyme activity in optimized medium composition achieves values between 135 and 140 IU/ml. The results suggest a promising model for recombinant production of PGA in B. megaterium with possible extracellular expression of the active enzyme.

Quinolinic acid catabolism is initiated by a novel four-component hydroxylase QuiA in Alcaligenes faecalis JQ191.

Quinolinic acid (QA) is an essential nitrogen-containing aromatic heterocyclic compounds in organisms and it also acts as an important intermediate in chemical industry, which has strong neurotoxicity and cytotoxicity. The wide range of sources and applications caused the release and accumulation of QA in the environment which might poses a hazard to ecosystems and human health. However, few research on the degradation of QA by microorganisms and toxicity of QA and its metabolites were reported. Alcaligenes faecalis JQ191 could degrade QA but the genetic foundation of QA degradation has not been studied. In this study, the gene cluster quiA1A2A3A4 was identified from A. faecalis JQ191, which was responsible for the initial catabolism step of QA. The quiA1A2A3A4 gene cluster encodes a novel cytoplasmic four-component hydroxylase QuiA. The 1H nuclear magnetic resonance indicated that QuiA catalyzed QA to 6-hydroxyquinolinic acid (6HQA) and the H218O-labeling analysis confirmed that the hydroxyl group incorporating into 6HQA was derived from water. Toxicity tests showed that the QA could approximately inhibit 20%-80% growth of Chlorella ellipsoidea, and 6HQA could relieve at least 50% QA growth inhibition of Chlorella ellipsoidea, indicating that the 6-hydroxylation of QA by QuiA is a detoxification process. This research provides new insights into the metabolism of QA by microorganism and potential application in the bioremediation of toxic pyridine derivatives-contaminated environments.

Biodegradation of low-density polyethylene (LDPE) through application of indigenous strain Alcaligenes faecalis ISJ128.

The resiliency of plastic products against microbial degradation in natural environment often creates devastating changes for humans, plants, and animals on the earth's surface. Biodegradation of plastics using indigenous bacteria may serve as a critical approach to overcome this resulting environmental stress. In the present work, a polyethylene degrading bacterium Alcaligenes faecalis strain ISJ128 (Accession No. MK968769) was isolated from partially degraded polyethylene film buried in the soil at plastic waste disposal site. The biodegradation studies were conducted by employing various methods such as hydrophobicity assessment of the strain ISJ128, measurement of viability and total protein content of bacterial biofilm attached to the polyethylene surface. The proliferation of bacterial cells on polyethylene film, as indicated by high growth response in terms of protein content (85.50 µg mL-1) and viability (1010 CFU mL-1), proposed reasonable suitability of our strain A. faecalis ISJ128 toward polyethylene degradation. The results of biodegradation assay revealed significant degradation (10.40%) of polyethylene film within a short period of time (i.e., 60 days), whereas no signs of degradation were seen in control PE film. A. faecalis strain ISJ128 also demonstrated a removal rate of 0.0018 day-1 along with half-life of 462 days. The scanning electron microscope (SEM) and Fourier transform infrared (FTIR) spectroscopy studies not only displayed changes on polyethylene surface but also altered level of intensity of functional groups and an increase in the carbonyl indexes justifying the degradation of polyethylene film due to bacterial activity. In addition, the secondary structure prediction (M fold software) of 16SrDNA proved the stable nature of the bacterial strain, thereby reflecting the profound scope of A. faecalis strain ISJ128 as a potential degrader for the eco-friendly disposal of polyethylene waste. Schematic representation of methodology.

Genomic and resistome analysis of Alcaligenes faecalis strain PGB1 by Nanopore MinION and Illumina Technologies.

BACKGROUND: Drug-resistant bacteria are important carriers of antibiotic-resistant genes (ARGs). This fact is crucial for the development of precise clinical drug treatment strategies. Long-read sequencing platforms such as the Oxford Nanopore sequencer can improve genome assembly efficiency particularly when they are combined with short-read sequencing data. RESULTS: Alcaligenes faecalis PGB1 was isolated and identified with resistance to penicillin and three other antibiotics. After being sequenced by Nanopore MinION and Illumina sequencer, its entire genome was hybrid-assembled. One chromosome and one plasmid was assembled and annotated with 4,433 genes (including 91 RNA genes). Function annotation and comparison between strains were performed. A phylogenetic analysis revealed that it was closest to A. faecalis ZD02. Resistome related sequences was explored, including ARGs, Insert sequence, phage. Two plasmid aminoglycoside genes were determined to be acquired ARGs. The main ARG category was antibiotic efflux resistance and ß-lactamase (EC 3.5.2.6) of PGB1 was assigned to Class A, Subclass A1b, and Cluster LSBL3. CONCLUSIONS: The present study identified the newly isolated bacterium A. faecalis PGB1 and systematically annotated its genome sequence and ARGs.

Genetic Foundations of Direct Ammonia Oxidation (Dirammox) to N2 and MocR-Like Transcriptional Regulator DnfR in Alcaligenes faecalis Strain JQ135.

Ammonia oxidation is an important process in both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox (direct ammonia oxidation, NH3→NH2OH→N2) has been identified in Alcaligenes ammonioxydans. However, whether Dirammox is present in other microbes, as well as its genetic regulation, remains unknown. In this study, it was found that the metabolically versatile bacterium Alcaligenes faecalis strain JQ135 could efficiently convert ammonia into N2 via NH2OH under aerobic conditions. Genetic deletion and complementation results suggest that dnfABC is responsible for the ammonia oxidation to N2 in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N2O and trace amounts of N2, with nitrite as the sole nitrogen source. Deletion of the nirK and nosZ genes, which are essential for denitrification, did not impair the capability of JQ135 to oxidize ammonia to N2 (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that pod (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and that AFA_16745 (which was previously annotated as ammonia monooxygenase and is widespread in heterotrophic bacteria) was not an ammonia monooxygenase. The MocR-family transcriptional regulator DnfR was characterized as an activator of the dnfABC operon with the binding motif 5'-TGGTCTGT-3' in the promoter region. A bioinformatic survey showed that homologs of dnf genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that, besides A. ammonioxydans, Dirammox occurs in other bacteria and is regulated by the MocR-family transcriptional regulator DnfR. IMPORTANCE Microbial ammonia oxidation is a key and rate-limiting step of the nitrogen cycle. Three previously known ammonia oxidation pathways (i.e., nitrification, anaerobic ammonia oxidation [Anammox], and complete ammonia oxidation [Comammox]) are mediated by autotrophic microbes. However, the genetic foundations of ammonia oxidation by heterotrophic microorganisms have not been investigated in depth. Recently, a previously unknown pathway, termed direct ammonia oxidation to N2 (Dirammox), has been identified in the heterotrophic bacterium Alcaligenes ammonioxydans HO-1. This paper shows that, in the metabolically versatile bacterium Alcaligenes faecalis JQ135, the Dirammox pathway is mediated by dnf genes, which are independent of the denitrification pathway. A bioinformatic survey suggests that homologs of dnf genes are widely distributed in bacteria. These findings enhance the understanding of the molecular mechanisms of heterotrophic ammonia oxidation to N2.

The TetR Family Repressor HpaR Negatively Regulates the Catabolism of 5-Hydroxypicolinic Acid in Alcaligenes faecalis JQ135 by Binding to Two Unique DNA Sequences in the Promoter of Hpa Operon.

5-Hydroxypicolinic acid (5HPA), an important natural pyridine derivative, is microbially degraded in the environment. Previously, a gene cluster, hpa, responsible for 5HPA degradation, was identified in Alcaligenes faecalis JQ135. However, the transcription regulation mechanism of the hpa cluster is still unknown. In this study, the transcription start site and promoter of the hpa operon was identified. Quantitative reverse transcription-PCR and promoter activity analysis indicated that the transcription of the hpa operon was negatively regulated by a TetR family regulator, HpaR, whereas the transcription of hpaR itself was not regulated by HpaR. Electrophoretic mobility shift assay and DNase I footprinting revealed that HpaR bound to two DNA sequences, covering the -35 region and -10 region, respectively, in the promoter region of the hpa operon. Interestingly, the two binding sequences are partially palindromic, with 3 to 4 mismatches and are complementary to each other. 5HPA acted as a ligand of HpaR, preventing HpaR from binding to promoter region and derepressing the transcription of the hpa operon. The study revealed that HpaR binds to two unique complementary sequences of the promoter of the hpa operon to negatively regulate the catabolism of 5HPA. IMPORTANCE This study revealed that the transcription of the hpa operon was negatively regulated by a TetR family regulator, HpaR. The binding of HpaR to the promoter of the hpa operon has the following unique features: (i) HpaR has two independent binding sites in the promoter of the hpa operon, covering -35 region and -10 region, respectively; (ii) the palindrome sequences of the two binding sites are complementary to each other; and (iii) both of the binding sites include a 10-nucleotide partial palindrome sequence with 3 to 4 mismatches. This study provides new insights into the binding features of the TetR family regulator with DNA sequences.

PicR as a MarR Family Transcriptional Repressor Multiply Controls the Transcription of Picolinic Acid Degradation Gene Cluster pic in Alcaligenes faecalis JQ135.

Picolinic acid (PA) is a natural toxic pyridine derivative as well as an important intermediate used in the chemical industry. In a previous study, we identified a gene cluster, pic, that responsible for the catabolism of PA in Alcaligenes faecalis JQ135. However, the transcriptional regulation of the pic cluster remains known. This study showed that the entire pic cluster was composed of 17 genes and transcribed as four operons: picR, picCDEF, picB4B3B2B1, and picT1A1A2A3T2T3MN. Deletion of picR, encoding a putative MarR-type regulator, greatly shortened the lag phase of PA degradation. An electrophoretic mobility shift assay and DNase I footprinting showed that PicR has one binding site in the picR-picC intergenic region and two binding sites in the picB-picT1 intergenic region. The DNA sequences of the three binding sites have the palindromic characteristics of TCAG-N4-CTNN: the space consists of four nonspecific bases, and the four palindromic bases on the left and the first two palindromic bases on the right are strictly conserved, while the last two bases on the right vary among the three binding sites. An in vivo ß-galactosidase activity reporter assay indicated that 6-hydroxypicolinic acid but not PA acted as a ligand of PicR, preventing PicR from binding to promoter regions and thus derepressing the transcription of the pic cluster. This study revealed the negative transcriptional regulation mechanism of PA degradation by PicR in A. faecalis JQ135 and provides new insights into the structure and function of the MarR-type regulator. IMPORTANCE The pic gene cluster was found to be responsible for PA degradation and widely distributed in Alpha-, Beta-, and Gammaproteobacteria. Thus, it is very necessary to understand the regulation mechanism of the pic cluster in these strains. This study revealed that PicR binds to three sites of the promoter regions of the pic cluster to multiply regulate the transcription of the pic cluster, which enables A. faecalis JQ135 to efficiently utilize PA. Furthermore, the study also found a unique palindrome sequence for binding of the MarR-type regulator. This study enhanced our understanding of microbial catabolism of environmental toxic pyridine derivatives.

The Novel Monooxygenase Gene dipD in the dip Gene Cluster of Alcaligenes faecalis JQ135 Is Essential for the Initial Catabolism of Dipicolinic Acid.

Dipicolinic acid (DPA), an essential pyridine derivative biosynthesized in Bacillus spores, constitutes a major proportion of global biomass carbon pool. Alcaligenes faecalis strain JQ135 could catabolize DPA through the "3HDPA (3-hydroxydipicolinic acid) pathway." However, the genes involved in this 3HDPA pathway are still unknown. In this study, a dip gene cluster responsible for DPA degradation was cloned from strain JQ135. The expression of dip genes was induced by DPA and negatively regulated by DipR. A novel monooxygenase gene, dipD, was crucial for the initial hydroxylation of DPA into 3HDPA and proposed to encode the key catalytic component of the multicomponent DPA monooxygenase. The heme binding protein gene dipF, ferredoxin reductase gene dipG, and ferredoxin genes dipJ/dipK/dipL were also involved in the DPA hydroxylation and proposed to encode other components of the multicomponent DPA monooxygenase. The 18O2 stable isotope labeling experiments confirmed that the oxygen atom in the hydroxyl group of 3HDPA came from dioxygen molecule rather than water. The protein sequence of DipD exhibits no significant sequence similarities with known oxygenases, suggesting that DipD was a new member of oxygenase family. Moreover, bioinformatic survey suggested that the dip gene cluster was widely distributed in many Alpha-, Beta-, and Gammaproteobacteria, including soil bacteria, aquatic bacteria, and pathogens. This study provides new molecular insights into the catabolism of DPA in bacteria. IMPORTANCE Dipicolinic acid (DPA) is a natural pyridine derivative that serves as an essential component of the Bacillus spore. DPA accounts for 5 to 15% of the dry weight of spores. Due to the huge number of spores in the environment, DPA is also considered to be an important component of the global biomass carbon pool. DPA could be decomposed by microorganisms and enter the global carbon cycling; however, the underlying molecular mechanisms are rarely studied. In this study, a DPA catabolic gene cluster (dip) was cloned and found to be widespread in Alpha-, Beta-, and Gammaproteobacteria. The genes responsible for the initial hydroxylation of DPA to 3-hydroxyl-dipicolinic acid were investigated in Alcaligenes faecalis strain JQ135. The present study opens a door to elucidate the mechanism of DPA degradation and its possible role in DPA-based carbon biotransformation on earth.

Complete genome analysis of the novel Alcaligenes faecalis phage vB_AfaP_QDWS595.

A novel lytic phage named vB_AfaP_QDWS595 infecting Alcaligenes faecalis was isolated and characterized in this study. The genome of phage vB_AfaP_QDWS595 was sequenced and analyzed, and the result revealed that the phage contained 70,466 bp of double-stranded DNA with 41.12% GC content. There were 74 putative genes encoding proteins as well as 11 tRNAs predicted in the phage genome. Phenotype and phylogeny analysis indicated that this phage might be a new member of the family Schitoviridae.

Constitutive secretory expression and characterization of nitrilase from Alcaligenes faecalis in Pichia pastoris for production of R-mandelic acid.

Nitrilases can directly hydrolyze nitrile compounds into carboxylic acids and ammonium. To solve the current problems of bioconversions using nitrilases, including the difficult separation of products from the resting cells used as the catalyst and high costs of chemical inducers, a nitrilase from Alcaligenes faecalis was heterologously expressed in Pichia pastoris X33. The stable nitrilase-expressing strain No.39-6-4 was obtained after three rounds of screening based on a combined detection method including dot-blot, SDS-PAGE, and western blot analyses, which confirmed the presence of recombinant nitrilase with a molecular mass of about 50 kDa. The temperature and pH optima of the nitrilase were 45°C and pH 7.5, respectively. Cu2+ , Zn2+ , and Tween 80 strongly inhibited the enzyme activity, but the optical purity of the product R-mandelic acid (R-MA) was stable, with practically 100% enantiomeric excess (ee). The nitrilase-producing P. pastoris strain developed in this study provides a basis for further research on the enzyme.

The efficient biomineralization and adsorption of cadmium (Cd2+) using secretory organo-biominerals (SOBs) produced by screened Alcaligenes faecalis K2.

Cadmium-contaminated wastewater has attracted increasing concerns due to its non-biodegradable properties and high toxicity. To explore eco-friendly and economically feasible strategies, the screened Alcaligenes faecalis K2 were employed for the biomineralization and recovery of Cd2+ from wastewater while producing considerable secretory organo-biominerals (SOBs) as bioadsorbents. At 75 mg/L Cd2+ exposure, 85.5% of Cd2+ was removed by K2, 43.0% of which was fixed in the granular SOBs. SOBs were convenient for separating from the solution. The adsorption capacity of granular sorbent made from SOBs was verified to be greater than 77.1 mg/g. Practically, 89.5% of 75 mg/L of Cd2+ could be stably removed while ereK2 continuously generated SOBs in a moving-bed biofilm reactor (MBBR). To sum up, the production of bioadsorbents can be achieved by K2, while removing Cd with live microorganisms, which was conducive to making full use of materials and improving Cd removal efficiency.

Coordinated binding of a two-component insecticidal protein from Alcaligenes faecalis to western corn rootworm midgut tissue.

AfIP-1A/1B is a two-component insecticidal protein identified from the soil bacterium Alcaligenes faecalis that has high activity against western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte). Previous results revealed that AfIP-1A/1B is cross-resistant to the binary protein from Bacillus thuringiensis (Bt), Cry34Ab1/Cry35Ab1 (also known as Gpp34Ab1/Tpp35Ab1; Crickmore et al., 2020), which was attributed to shared binding sites in WCR gut tissue (Yalpani et al., 2017). To better understand the interaction of AfIP-1A/1B with its receptor, we have systematically evaluated the binding of these proteins with WCR brush border membrane vesicles (BBMVs). Our findings show that AfIP-1A binds directly to BBMVs, while AfIP-1B does not; AfIP-1B binding only occurred in the presence of AfIP-1A which was accompanied by the presence of stable, high molecular weight oligomers of AfIP-1B observed on denaturing protein gels. Additionally, we show that AfIP-1A/1B forms pores in artificial lipid membranes. Finally, binding of AfIP-1A/1B was found to be reduced in BBMVs from Cry34Ab1/Cry35Ab1-resistant WCR where Cry34Ab1/Cry35Ab1 binding was also reduced. The reduced binding of both proteins is consistent with recognition of a shared receptor that has been altered in the resistant strain. The coordination of AfIP-1B binding by AfIP-1A, the similar structures between AfIP-1A and Cry34Ab1, along with their shared binding sites and cross-resistance, suggest a similar role for AfIP1A and Cry34Ab1 in receptor recognition and docking site for their cognate partners, AfIP-1B and Cry35Ab1, respectively.

Heterotrophic nitrification and related functional gene expression characteristics of Alcaligenes faecalis SDU20 with the potential use in swine wastewater treatment.

A new heterotrophic nitrifying bacterium was isolated from the compost of swine manure and rice husk and identified as Alcaligenes faecalis SDU20. Strain SDU20 had heterotrophic nitrification potential and could remove 99.7% of the initial NH4+-N. Nitrogen balance analysis revealed that 15.9 and 12.3% of the NH4+-N were converted into biological nitrogen and nitrate nitrogen, respectively. The remaining 71.44% could be converted into N2 or N2O. Single-factor experiments showed that the optimal conditions for ammonium removal were the carbon source of sodium succinate, C/N ratio 10, initial pH 8.0, and temperature 30 °C. Nitrification genes were determined to be upregulated when sodium succinate was used as the carbon source analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Strain SDU20 could tolerate 4% salinity and show resistance to some heavy metal ions. Strain SDU20 removed 72.6% high concentrated NH4+-N of 2000 mg/L within 216 h. In a batch experiment, the highest NH4+-N removal efficiency of 98.7% and COD removal efficiency of 93.7% were obtained in the treatment of unsterilized swine wastewater. Strain SDU20 is promising in high-ammonium wastewater treatment.

Extraction of chitin from Litopenaeus vannamei shell and its subsequent characterization: an approach of waste valorization through microbial bioprocessing.

Chemical extraction of chitin is very hazardous and costly which can be overwhelmed by microbial bioprocessing. In this study, potent protease and lactic acid-producing bacteria were screened and identified as Alcaligens faecalis S3 and Bacillus coagulans L2, respectively. Productions of protease and lactic acid by the respective bacterial strains were optimized. The shell of Litopenaeus vannamei was sequentially treated with the partially purified protease and lactic acid and the treatment conditions were optimized for betterment of chitin yield. Spectral characterization by SEM-EDS, IR, XRD, NMR, XPS and thermal characterization by TG and DTG analysis of the extracted chitin was made and compared with commercial one. It was revealed that both the chitin have similar characteristics. Therefore, it can be articulated that chitin can be extracted from crustacean shells in pure form by microbial bioprocessing which will be a good catch for biorefinary industries for chitin extraction through greener route.

Simultaneous removal characteristics of ammonium and phenol by Alcaligenes faecalis strain WY-01 with the addition of acetate.

In this study, simultaneous removal of ammonium plus phenol could be achieved by Alcaligenes faecalis strain WY-01 with the addition of acetate, although acetate delayed the phenol degradation, probably due to the delayed expression of phenol hydroxylase gene under the presence of acetate. Moreover, the successful expression of key enzyme genes in strain WY-01 provided some evidence to illustrate its metabolic pathways of ammonium and phenol under aerobic conditions. Furthermore, SEM was used to clarify the role of acetate in resisting phenol toxicity, and these results demonstrated that strain WY-01 has the ability to form cell flocs when sodium acetate is used as co-substrate for a high concentration of phenol, and these flocs could protect cells against the toxicity of phenol, further enhancing phenol degradation in a high concentration of phenol. All these will provide further insights into the efficacy of strain WY-01 for treating wastewater cocontaminated by ammonium and phenol.