Novel OBSL1 Variant in a Chinese Patient with 3M Syndrome and the c.458dupG Mutation May Be a Potential Hotspot Mutation in the Chinese Population.

3M syndrome is an autosomal recessive disorder characterized by short stature and skeletal developmental abnormalities. In this study, a Chinese patient with 3M syndrome was presented. A novel OBSL1 (obscurin-like 1 gene) variant was found. The patient is a 2-year-old girl who presented with short stature and had intrauterine growth retardation and low birth weight. Gene analysis revealed compound heterozygote mutations in the OBSL1 gene: c.458dupG (p.L154Pfs*100) and c.427dupG (p.A143Gfs*111). The c.427dupG mutation is novel. The c.458dupG mutation has been documented in 5 cases, occurring only in Chinese individuals, indicating ethnic specificity. In cases of short-statured children presenting intrauterine growth retardation, low birth weight, and skeletal developmental abnormalities, 3M syndrome should be considered. The c.458dupG mutation might be a hotspot mutation in the Chinese population.

A synonymous KCNJ11 variant leading to MODY13: A case report and literature review.

Background: Maturity-onset diabetes of the young, type 13 (MODY13) is a specific subclass of monogenic diabetes mellitus that does not exhibit the typical clinical manifestations of diabetes, necessitating the use of genetic testing for accurate diagnosis. With the progression of monogenic diabetes and MODY, the number of reported MODY13 cases has reached a minimum of 22. Nevertheless, there remains a dearth of information regarding patients diagnosed with MODY13 presenting synonymous variants. Case presentation: This study presents a description of the clinical and genetic features of a 9-year-old male patient diagnosed with MODY13. A noteworthy finding in this case was the occurrence of a "separation phenomenon" between C-peptide and insulin during the standard meal test. Whole exome sequencing (WES) identified a KCNJ11 c.843C > T (p.L281=) mutation in exon 1, which contradicted the previously reported phenotype. Following the onset of ketosis, the patient underwent insulin therapy for a duration of one month, during which the insulin dosage was gradually modified based on blood glucose levels. In order to maintain normoglycemia, he adhered to a diabetic dietary regimen and participated in 1-2 h of moderate exercise daily. Conclusion: The study implies that patient with KCNJ11 variant shows a "separation phenomenon" between C-peptide and insulin in standard meal test. Our report also enriched the genotype and phenotype spectrums of MODY13 and highlighted the importance of genetic testing in patients without characteristic clinical symptoms of diabetes.

Nanoscale zero-valent iron alleviate antibiotic resistance risk during managed aquifer recharge (MAR) by regulating denitrifying bacterial network.

The frequent occurrence of antibiotics in reclaimed water is concerning, in the case of managed aquifer recharge (MAR), it inevitably hinders further water purification and accelerates the evolutionary resistance in indigenous bacteria. In this study, we constructed two column reactors and nanoscale zero-valent iron (nZVI) amendment was applied for its effects on water quality variation, microbial community succession, and antibiotic resistance genes (ARGs) dissemination, deciphered the underlying mechanism of resistance risk reduction. Results showed that nZVI was oxidized to iron oxides in the sediment column, and total effluent iron concentration was within permissible limits. nZVI enhanced NO3--N removal by 15.5% through enriching denitrifying bacteria and genes, whereas made no effects on oxacillin (OXA) removal. In addition, nZVI exhibited a pivotal impact on ARGs and plasmids decreasing. Network analysis elucidated that the diversity and richness of ARG host declined with nZVI amendment. Denitrifying bacteria play a key role in suppressing horizontal gene transfer (HGT). The underlying mechanisms of inhibited HGT included the downregulated SOS response, the inhibited Type-Ⅳ secretion system and the weakened driving force. This study afforded vital insights into ARG spread control, providing a reference for future applications of nZVI in MAR.

Exploring an Efficient and Eco-Friendly Signaling Molecule and Its Quorum Quenching Ability for Controlling Microcystis Blooms.

Globally, cyanobacterial blooms have become serious problems in eutrophic water. Most previous studies have focused on environmental factors but have neglected the role of quorum sensing (QS) in bloom development and control. This study explored a key quorum sensing molecule (QSM) that promotes cell growth and then proposed a targeted quorum quencher to control blooms. A new QSM 3-OH-C4-HSL was identified with high-resolution mass spectrometry. It was found to regulate cellular carbon metabolism and energy metabolism as a means to promote Microcystis aeruginosa growth. To quench the QS induced by 3-OH-C4-HSL, three furanone-like inhibitors were proposed based on molecular structure, of which dihydro-3-amino-2-(3H)-furanone (FN) at a concentration of 20 µM exhibited excellent inhibition of M. aeruginosa growth (by 67%). Molecular docking analysis revealed that the inhibitor strongly occupied the QSM receptor protein LuxR by binding with Asn164(A) and His167(A) via two hydrogen bonds (the bond lengths were 3.04 and 4.04 Å) and the binding energy was -5.9 kcal/mol. The inhibitor blocked signaling regulation and induced programmed cell death in Microcystis. Importantly, FN presented little aquatic biotoxicity and negligibly affected aquatic microbial function. This study provides a promising new and eco-friendly strategy for controlling cyanobacterial blooms.

Selective pressure of PFOA on microbial community: Enrichment of denitrifiers harboring ARGs and the transfer of ferric-electrons.

Perfluorooctanoic acid (PFOA), a class of permanent organic pollutants, is frequently detected in surface and ground water, with the latter made up primarily of porous media (such as soils, sediments, and aquifers) that harbor microbial communities. Therefore, we investigated the effects of PFOA on water ecosystems and found that, under stimulation by 2.4 µM PFOA, denitrifiers were significantly enriched due to their hosting antibiotic resistant genes (ARGs), which were 1.45 times more abundant than the control. Furthermore, denitrifying metabolism was enhanced by Fe(II) electron donation. Specifically, 2.4 µM PFOA significantly enhanced the removal of total inorganic nitrogen by 178.6%. The microbial community became predominated by denitrifying bacteria (67.8% abundance). Notably, the nitrate-reduction ferrous-oxidizing (NRFO) bacteria Dechloromonas, Acidovorax, Bradyrhizorium, etc. were significantly enriched. The selective pressures of PFOA driving the enrichment of denitrifiers were twofold. First, the toxic PFOA induced denitrifying bacteria to produce ARGs, mainly including the efflux (occupying 55.4%) and antibiotic inactivation (occupying 41.2%) types, which improved microbial tolerance to PFOA. The risk of horizontal ARGs transmission was elevated as the overall number of horizontally transmissible ARGs increased by 47.1%. Second, Fe(II) electrons were transported via the porin-cytochrome c extracellular electrons transfer system (EET), promoting the expression of nitrate reductases, which in turn further enhanced denitrification. In summary, PFOA regulated the microbial community structure and influenced microbial TN removal functions and increased the contribution of ARGs by the denitrifier hosts, but the PFOA-induced production of ARGs may pose a serious ecological threat that needs to be comprehensively investigated.

Accumulation of nitrite after reclaimed water recharge due to the disinfection byproduct chlorite.

Due to its toxicity, the disinfection byproduct chlorite in drinking water is strictly regulated to be ≤ 1.0 mg/L, but in reclaimed, non-drinking water chlorite is unregulated and rarely considered. However, chlorite is cytotoxic and has a high oxidation potential. Therefore, as reclaimed water infiltrates soil and groundwater, it may alter the soil environment and microbial community, which may affect the degradation of organic matter and the transformation of the N element. In this study, the effects of reclaimed water containing chlorite on soil microorganisms were investigated by simulating subsurface infiltration. It was found that chlorite improved the conversion of nitrate nitrogen to nitrite nitrogen, but inhibited further conversion of nitrite nitrogen. The nitrite nitrogen in the effluent reached 4.61 mg/L when chlorite was present, while only 0.16 mg/L was found in the control system. The chlorite produced obvious oxidative stress reactions in cells, inhibited the EPSs production, in which the contents of polysaccharides and proteins reduced by nearly 41% and 62%, respectively. Besides, chlorite resulted in the enrichment of efflux resistance genes in the microbial community, mainly adeF and cmlB1. Self-protection against chlorite is achieved mainly using efflux pump related genes. Metagenomics data analysis showed that Delftia became the dominant genus when exposed to chlorite, with the greatest abundance at 17.9%. Chlorite also resulted in the upregulated expression of nar genes (by more than 149%) and downregulation of nir gene expression (by more than 62%). This study reveals the effects of the disinfection byproduct chlorite on a soil microecosystem, providing important information for the management and reuse of reclaimed water.

Case Report: Recombinant human growth hormone therapy in a patient with spondyloepiphyseal dysplasia, Kondo-Fu type.

Background: Variants in membrane-bound transcription factor peptidase, site 1 (MBTPS1) gene, can result in clinically rare spondyloepiphyseal dysplasia of Kondo-fu type (OMIM #618392, SEDKF), Silver-Russell syndrome, and CAOP (cataract, alopecia, oral mucosal disorder, and psoriasis-like) syndrome. Case presentation: A 6-year-old Chinese male child diagnosed with SEDKF underwent 3 years of growth hormone therapy. A genetic examination revealed two new nonsense variants in the MBTPS1 gene on chromosome 16q23-q24 with compound heterozygotes c.1589(exon12)A > G and c.163(exon2)G > A. Conclusion: The MBTPS1 gene c.1589(exon12)A > G and c.163(exon2)G > A on chromosome 16q23-q24 is associated with SEDKF. Growth hormone therapy can repair growth retardation in patients with spondyloepiphyseal dysplasia, Kondo-Fu type; however, more evidence of such patient cases is required to support this hypothesis.

Self-rescue of nitrogen-cycling bacteria under ß-lactam antibiotics stress during managed aquifer recharge (MAR): Microbial collaboration and anti-resistance.

Storing reclaimed water via managed aquifer recharge (MAR) is an effective strategy for alleviating groundwater overdraft and achieving water resource recycling simultaneously. However, ß-lactam antibiotics in the reclaimed water can induce stress on aquifer system, reshape microbial community, and affect the emergence and prevalence of antibiotic resistance genes (ARGs). In this study, three sandy soil columns (H 1.5 m, ID 14 cm) were employed to simulate MAR, and synthetic reclaimed water containing either amoxicillin (AMO), ampicillin (AMP) or oxacillin (OXA) was continuously recharged for 120 d The temporal and spatial attenuation of ß-lactams and nitrogen was studied, and microbial collaboration and the resistance mechanism were elaborated. Biodegradation is the main pathway for ß-lactams elimination, AMO and AMP were eliminated when migrating 30 cm, while the attenuation of OXA experienced in the whole column with final removal efficiency of 82%. Moreover, refractory OXA induced more ARGs production, and approximately 10% and 13% higher than that of AMO and AMP columns. Efflux pump and antibiotics inactivation were the two major resistance mechanisms. NO3--N gradually decreased (by 26%, 38%, and 49% for AMO, AMP, and OXA, respectively) along the recharge direction. Microbial co-occurrence network revealed that nitrogen-cycling bacteria were the keystone species in aquifer community, and ammonation provided NH4+-N for the nitrification process of ammonia-oxidizing archaea (AOA), promoting the further denitrification for nitrogen removal in MAR process. Nitrogen-cycling bacteria were the key and active ARG hosts, which could keep nitrogen transformation activity under antibiotics stress. In sum, nitrogen-cycling bacteria exhibited intimate collaboration and elastic resistance in response to the malnutrition environment and ß-lactams exposure during MAR.

Effects of trace PFOA on microbial community and metabolisms: Microbial selectivity, regulations and risks.

Perfluorooctanoic acid (PFOA), a "forever chemical", is continuously discharged and mitigated in the environment despite its production and use being severely restricted globally. Due to the transformation, attachment, and adsorption of PFOA in aquatic environments, PFOA accumulates in the porous media of sediments, soils, and vadose regions. However, the impact of trace PFOA in the porous media on interstitial water and water safety is not clear. In this work, we simulated a porous media layer using a sand column and explored the effects of µg-level PFOA migration on microbial community alternation, microbial function regulation, and the generation and spread of microbial risks. After 60 days of PFOA stimulation, Proteobacteria became the dominant phylum with an abundance of 91.8%, since it carried 71% of the antibiotic resistance genes (ARGs). Meanwhile, the halogen-related Dechloromonas abundance increased from 0.4% to 10.6%. In addition, PFOA significantly stimulated protein (more than 1288%) and polysaccharides (more than 4417%) production by up-regulating amino acid metabolism (p< 0.001) and membrane transport (p < 0.001) to accelerate the microbial aggregation. More importantly, the rapidly forming biofilm immobilized and blocked PFOA. The more active antioxidant system repaired the damaged cell membrane by significantly up-regulating glycerophospholipid metabolism and peptidoglycan biosynthesis. It is worth noting that PFOA increased the abundance of antibiotic resistance genes (ARGs) and human bacterial pathogens (HBPs) in porous media by 30% and 106%. PFOA increased the proportion of vertical transmission ARGs (vARGs), and co-occurrence network analysis (r ≥ 0.8, p ≤ 0.01) verified that vARGs were mainly mediated by HBPs. A comprehensive understanding of PFOA interactions with its microecological environment is provided.

Transboundary intercellular communications between Penicillium and bacterial communities during sludge bulking: Inspirations on quenching fungal dominance.

Fungal bulking is caused by the evolution toward a fungi-dominant unbalanced sludge system, which is indeed the phenomenon of fungi competing against bacterial cells. We hypothesized that the cross-kingdom intercellular communication between fungi and bacteria was internal driving force that stimulated fungal bulking. In this study, we identified three signal molecules related to Penicillium fungi bulking under low-pH stress in an activated sludge reactor, which inspired us to propose a sludge bulking prevention strategy using the quorum quenching theory. When pH dropped from 7.0 to 4.5, the abundance of Penicillium increased from 12.5% to 44.8%. However, some functional bacterial genera, such as Nitrosomonas and Sphingopyxis, were washed out from the sludge. The production of quorum-sensing (QS) molecules N-Heptanoyl-L-homoserine lactone (C7-HSL), N-Dodecanoyl-L-homoserine lactone (C12-HSL), and N-Tetradecanoyl-L-homoserine lactone (C14-HSL) was regulated with sludge bulking; especially the response of the latter two was significantly negative to Penicillium blooming (P < 0.05). To test their roles, trace commercial C12-HSL and C14-HSL were added to Penicillium culture, successfully causing 8.3% and 30.2% inhibition of mycelial formation, respectively. They also contributed to the improvement of activated sludge settleability by 6.1% and 39.7%, respectively (represented by sludge volume index). The transcriptome technique further revealed the regulation of the expression of genes in |logFC| >1, involving signal transduction, mycelium synthesis, and metabolic pathways. Our study provided an innovative strategy for controlling fungal bulking from the perspective of microbial transboundary informatics.

C12-HSL is an across-boundary signal molecule that could alleviate fungi Galactomyces's filamentation: A new mechanism on activated sludge bulking.

Fungal bulking is caused by fungi excessive growth and morphological changes, resulting from the evolution toward fungi dominant activated sludge. Communication across fungi and bacteria boundary that mediated by bacterial signal molecules (SMs) probably is the central induce caused fungal bulking occurrence. In this work, it intended to identify the bacterial SM that affected fungal bulking, and verified its roles in regulate the spore germination and hyphal growth. We found C12-HSL concentration decreased significantly from 12.36 to 3.38 ng/g-VSS (P < 0.05) when fungal sludge bulking happened, and filamentous Galactomyces's relatively abundant was correlatively enriched. To test the effects of this SM, trace commercial C12-HSL was added to pure cultured Galactomyces, in which spore germination rates decreased by 20 % and hyphal extension inhibited by 15 %. Ras1-cAMP-PKA and mitogen-activated protein kinase (MAPK) pathways of Galactomyces were responsible for signal C12-HSL transduction, which inhibited peroxisome biosynthesis, suppressed the biological activity of the actin cytoskeleton, and disrupted intercellular organelle transport. All these results showed C12-HSL was the functional SM that could suppress the development of fungal filamentous. This study provided a new insight into the sludge bulking mechanism from view of cross-kingdom communication.

Phosphomannomutase 2 hyperinsulinemia: Recent advances of genetic pathogenesis, diagnosis, and management.

Congenital hyperinsulinemia (CHI), is a clinically heterogeneous disorder that presents as a major cause of persistent and recurrent hypoglycemia during infancy and childhood. There are 16 subtypes of CHI-related genes. Phosphomannomutase 2 hyperinsulinemia (PMM2-HI) is an extremely rare subtype which is first reported in 2017, with only 18 families reported so far. This review provides a structured description of the genetic pathogenesis, and current diagnostic and therapeutic advances of PMM2-HI to increase clinicians' awareness of PMM2-HI.

C.487C>T mutation in PAX4 gene causes MODY9: A case report and literature review.

RATIONALE: Maturity-onset diabetes of the young (MODY) is a group of autosomal dominant monogenic diabetes mellitus with a wide range of clinical manifestations that require distinct treatment strategies. MODY9 (OMIM # 612225) is a rare type of MODY, caused by a mutation in the Paired box gene 4 (PAX4). PATIENT CONCERN: A 19-months boy was admitted to the department of endocrinology at Beijing Children's Hospital due to excessive water drinking, polyuria for over half a month, and wheezing for 3 days. DIAGNOSE: The whole-exon sequencing analysis demonstrated that the child carried the heterozygous missense mutation of c.487>T in the 7th exon region of PAX4 gene and diagnosed MODY9. INTERVENTION: The patient was treated with fluid therapy, ketosis correction, insulin, and anti-infection treatment. OUTCOMES: After 17 days in the hospital, the blood glucose levels remained stable and the patient was discharged. LESSONS: In Chinese children, the heterozygous mutation of c.487C>T in the PAX4 gene can lead to the occurrence of MODY9.Gene sequencing analysis is of great significance in the diagnosis and classification of MODY.

Carbon dots enhance the recovery of microalgae bioresources from wastewater containing amoxicillin.

The cultivation of microalgae using wastewater could bring some major economic benefits; however, the toxics in wastewater typically lead to a reduction in bioresource production. In this study, carbon dots (CDs) could enhance the photosynthetic activity of Chlorella under antibiotic stress because they might optimize photoluminescence by red-shifting incident light. Adding of 1 mg/L CDs increased the specific growth rate of Chlorella by 36.0% (day 8-13) and 52.7% (day 14-18) and significantly increased photosystems II activity. This treatment also increased amoxicillin removal by 18.6%. Thus, the toxicity of residuals was significantly eliminated (P < 0.05). The removal of nitrogen and phosphorous was increased by 14.6% and 9.9%, respectively. The production of pigments, lipids and proteins was increased by 16.6%, 19.5% and 24.8%, respectively. This work provided a new strategy of using CDs to mediate the coupling of microalgal bioresources production and toxic wastewater purification.

Benzoic and salicylic acid are the signaling molecules of Chlorella cells for improving cell growth.

Cell-to-cell communication regulates microalgae production via signaling molecules (SMs), but few microalgal SM species are known. Here, we document two new microalgae SMs, benzoic acid (BA) and salicylic acid (SA). Initially, crude SMs were extracted from a microalgae culture in which microalgae grew on heterotrophic-enriched phosphorus nutrition. The extracted SMs enhanced Chlorella growth by ∼72%, promoted nutrient uptake, and up-regulated the mitogen-activated protein-kinase signaling cascade. Fourier transform infrared and nuclear magnetic resonance analyses identified the putative SMs was aromatic carboxylic acids. BA and SA were identified using high-resolution mass spectrometry. BA and SA addition increased cell growth by ∼75% and ∼25%; and improved ATP production by ∼35% and ∼20%. Transcriptomic analysis showed that BA and SA were biosynthesized via CoA-dependent, non-oxidative pathway. The SMs upregulated TCA-cycle enzymes, which promoted carbon assimilation and activated DNA-replicating enzyme, so that accelerated cell division. This study identified two new SMs for microalgae cell communication and provides means to identify other SMs.

Co-substrate addition accelerated amoxicillin degradation and detoxification by up-regulating degradation related enzymes and promoting cell resistance.

ß-Lactam antibiotics are the most commonly used antibiotics, and are difficult to remove by conventional biological treatments because of their persistent and toxic nature. The addition of co-substrates has been successfully employed to improve the removal of refractory pollutants. So, we hypothesized that the co-substrate strategy would increase antibiotic degradation and benefit microbial survival. In this work, we reported that co-substrate (acetate) addition up-regulated key degrading enzymes and resistance related genes in a model bacteria strain (L. aquatilis) when being treated with 0.055 mM amoxicillin (AMO). ß-Lactamase, amidases, transaminase, and amide C-N hydrolase showed increased activation. As a result, AMO removal reached ∼95 %, a ∼60 % increase over the control. Furthermore, the addition of acetate drove the down-stream TCA cycle, which accelerated the detoxification of the intermediates and reduced the microbial inhibition by the antibiotic products to as low as ∼15 %. Besides, the expression levels of genes encoding the efflux pump, penicillin binding proteins, and ß-Lactamase were up-regulated, and the inhibition of peptidoglycan biosynthesis was down-regulated. The cell density was enhanced by ∼170 % and showed improved DNA replication. In conclusion, the addition of the co-substrate accelerated AMO degradation and detoxification by up-regulating degrading enzymes and promoting cell resistance.

Eliminating partial-transformation products and mitigating residual toxicity of amoxicillin through intimately coupled photocatalysis and biodegradation.

Intimately coupled photocatalysis and biodegradation (ICPB) is a promising technology for treating wastewater containing antibiotics. While past work has documented the benefits of ICPB for removing and mineralizing antibiotics, its impacts on mitigating biotoxicity from products has not been studied. We fabricated an ICPB carrier by coating Ag-doped TiO2 on the outer skeleton of sponge carriers and allowing biofilm to grow in the internal macro-pores. We used amoxicillin (C16H19N3O5S) as the model antibiotic. The amoxicillin-removal rate contents with ICPB was greater by 40% vs. photocatalysis and 65% vs. biodegradation, based on the first-order kinetic simulation. While mineralization of amoxicillin was minimal for photocatalysis or biodegradation alone, it was ∼35% with ICPB. Photocatalysis alone led to accumulation of C14H21N3O2S; biodegradation alone resulted in accumulation of C14H21N3O3, C16H18N2O4S, and C15H21N3O3; but they were negligible after ICPB. As a result, ICPB reduced toxicity impacts measured by Staphylococcus aureas growth, Daphnia magna mobility, and teratogenicity to Zebrafish embryos. In contrast, photocatalysis alone increased each of the toxicity effects. In sum, ICPB gave greater removal and mineralization of amoxicillin, and it mitigated biotoxicity from treatment products.

Exogenous refractory protein enhances biofilm formation by altering the quorum sensing system: A potential hazard of soluble microbial proteins from WWTP effluent.

Soluble microbial refractory proteins are major components of effluent from wastewater treatment plants that utilize a biological wastewater treatment process. The remaining proteins could negatively affect downstream treatment processes by altering the bacterial quorum sensing system. In this work, we elaborated the effects of exogenous refractory protein on biofilm formation. The results showed a linear relationship between biofilm formation and experimental protein concentrations at the range typically found in effluent, 0-8.0 mg/L. Micro-observation revealed that the exogenous refractory protein stimulated extracellular polysaccharide secretion to promote biofilm maturation. Extracellular polysaccharides increased by ~200% with the addition of only 2.0 mg/L protein. In addition, exogenous refractory proteins altered the quorum sensing system gene expression and polysaccharide gene expression. This work found that exogenous protein accelerated biofilm formation by influencing the quorum sensing system, thus providing new insight into the potential harm of soluble microbial refractory products.

Acceleration of saturated porous media clogging and silicon dissolution due to low concentrations of Al(III) in the recharge of reclaimed water.

The recharge of reclaimed water is an effective strategy for addressing the issues of water quality deterioration and groundwater level decline simultaneously. Residual Al coagulants are normally remained in the recovered water at low concentrations, and may induce clogging problems during the recharging process. However, this issue has been ignored in the past. In this study, we investigated the mechanisms of Al(III)-induced aquifer bio-clogging, the role of Al(III) in quartz sand media (SiO2) dissolution and re-precipitation in the series of aquifer columns. We determined that Al(III) resulted in serious clogging in ∼140 h at low concentrations that satisfied the national drinking water standard of China. The corresponding hydraulic conductivity decreased by more than ∼90% in the bacteria-containing aquifer, which was ∼30% greater than that for the bacteria-free trials. The enhanced Al(III)-related clogging was caused by modifying quartz sand to form Si-O-Al(OH)n and improving microbes attachment. Microbes retention kinetic coefficients (k) of the Al recharged simulated aquifer could increase by 3.0-8.3 times. The Al(III) also enhanced biomass production and clogging by binding to microbial extracellular polymeric substances. In turn, the greater amount of biomass accelerated the Si dissolution and re-precipitation, this may potentially damage the stability of aquifer structure. The results showed that reclaimed water treated with Al coagulation should be employed with caution for recharging.