an unexpected contaminant in our aquatic and costal environments is artificial sweeteners . due to the human inability to metabolize them naturally the highest concentration of artificial sweetener contaminants is in waste water treatment plants ' reservoirs . artificial sweeteners such as saccharin and cyclamates are detected in lower concentrations and are found 90% degraded by the wastewater treatment process . degradation only occurs to a limited extent during hydrolysis , ozonation , and microbial processes indicating that breakdown of sucralose will likely be slow and incomplete leading to accumulation of sucralose in surface waters . this indicates that conventional waste water treatment is ineffective at degrading sucralose . from wastewater facilities the pollutants are dumped into public waterways , and sucralose has been detected in rivers in north carolina , in the gulf stream , and even in the waters of the florida keys . also for the first time scientists are detecting sucralose in the usa inland surface waters . artificial sweeteners have been considered contaminants by environmental scientists for a short time because artificial sweeteners are water contaminants that are highly specific to wastewater . most artificial sweeteners are either partially or completely broken down due to the waste water treatment process using high temperatures , changes in ph , and constant filtration . it would seem that the exception is sucralose due to its ability to withstand drastic ph and temperature changes ; it is also small enough to pass through the filtration process associated with the treatment . hence , sucralose is being continuously released into our environment in ever increasing concentrations , and due to the human consumers ' inability to metabolize this artificial sweetener , we are dumping it into our water ways and it is collecting over time . as time passes sucralose will spread to other aquatic and costal ecosystems , increasing concentration . the persistent qualities of sucralose may lead to chronic low - dose exposure with largely unknown consequences for human and environmental health . it is also unknown what this increasing concentration of sucralose is doing to environmental microbes . studies of human oral and gut bacteria have shown an inhibition of bacterial growth in the presence of sucralose . in one study the incorporation of 126  mmol / l sucralose into glucose agar medium caused total inhibition of growth of streptococcus sobrinus 6715 - 17 , streptococcus sanguis 10904 , streptococcus sanguis challis , streptococcus salivarius , and actinomyces viscosus wvu627 . in a related study rats were infected with streptococcus sobrinus and given sucrose water diet , and they developed caries lesions . then another group of rats was given the same bacteria but sucralose water instead of sugar water . those rats had a drastic decrease in caries lesions in their teeth , demonstrating that oral bacteria can not grow on the artificial sweetener hence causing less damage , proving that sucralose is noncariogenic . these are good examples of sucralose inhibiting bacterial growth ; however , there have been minimal studies on the inhibitory effects of sucralose on environmental microbes . sucralose is increasing in concentration in our waterways , and it has been shown in previous studies to be harmful to oral and gut bacteria . water samples and soil samples from various test sites around jacksonville florida ( table 1 ) were collected aseptically using autoclaved collection flasks and jars . fluid and soil extracted from samples were serially diluted with sterile 0.89%  nacl solution , then spread plated on tryptic soy agar ( tsa ) ( difco laboratories , michigan , usa ) plates infused with 80  mm sucralose , and incubated at 32.7c for 48  h. bacteria were isolated into pure cultures on subsequent tsa slants based on colony morphology ( table 2 ) . isolates of each bacterium were incubated ( 32.7c ) for one day for microbial analysis . the isolates were then analyzed using basic light microscopy to identify individual gram characters and cellular morphology ( table 2 ) . once a list of the isolates with their microbial characters was formed , they were screened for sucralose metabolism . in order to inspect organismal growth in the presence of sucralose , 0.1  ml of isolated cell cultures was diluted with 2.9  ml of 0.89% nacl solution . these samples were streak plated onto m9 agar containing glucose ( technova , nova scotia , canada ) ( positive control ) , m9 agar containing sucrose ( positive control ) , m9 agar containing sucralose and glucose ( experimental ) , m9 agar containing sucralose ( experimental ) , and m9 agar containing no sugars ( negative control ) . isolates which exhibited growth on the m9 agar containing sucralose and glucose were selected for further experimentation ( table 3 ) . this is due to the possibility that they may be resistant to or metabolizing sucralose , which was inspected during the growth testing experiment . genomic dna was extracted from each of the selected bacterial isolates using the ultraclean microbial dna isolation kit in accordance with the manufacturer protocols ( mo bio laboratories , california , usa ) . the 16s rrna gene was amplified using the bacterial consensus primers 8f ( 5 agttgatcctggctcag 3 ) and 1492r ( 5 accttgttacgactt 3 ) . the long polymerase chain reactions ( pcr ) consisted of 41.7  l dh2o , 5.0  l 10x taq buffer , 1.5  l 50  mm  mgcl2 , 0.5  l 10  m forward primer 10  m reverse primer 0.4  l 25  mm dntps , 0.4  l 5  u/l taq polymerase , and 1  l genomic dna in a final volume of 50  l . dna amplification was performed with the following thermocycler regime : 2  min at 98c followed by 33 cycles of , 98c for 30  s , 45c for 60  s , 72c for 90  s and a single step at 72c for 10  min . short pcr amplification consisted of 50  l reactions with analogous reagents / concentrations to the long pcr , using the additional primers 760r ( 5 ctaccagggtatctaat 3 ) and 790f ( 5 attagataccctggtag 3 ) with the following thermocycler settings : 25 cycles of 98c for 30  s , 44c for 45  s , and 72c for 90  s.   the short pcr products were cleaned up using the qiaquick pcr purification kit following the manufacturer protocols ( qiagen , california , usa ) . the four primers aforementioned were employed for cycle sequencing on a ceq 8000 genetic analysis system ( beckman coulter , california , usa ) using 1  l genomelab dtcs quick start master mix , 2  l primer , 2  l dna , and 7  l dh2o . cycle sequencing consisted of 33 cycles at 96c for 30  s , 3747c for 15  s , and 60c for 4  min . sequencing reactions were performed using each of the amplification primers and internal primers so that each fragment was sequenced in both the forward and reverse directions . products were cleaned and precipitated according to the manufacturer specifications ( beckman coulter , california , usa ) . the obtained sequences were compared to other sequences using the blast function through the ncbi website ( http://www.ncbi.nlm.nih.gov/blast/ ) . individual isolates were then cultured in tryptic soy broth ( tsb ) media ( difco laboratories , michigan , usa ) and incubated at 25f . the control group consisted of 5  ml of tsb , and the experimental groups included 5  ml tsb with 0.5  ml of 10% , 20% , 30% , and 40% sucralose added , respectively . turbidity of the cultures was measured over the next 9 days at the same time each day using a sequoia turner ultraviolet light spectrophotometer set to 620  nm wavelength ( figure 1 ) . individual isolates were cultured in m9 broth media ( technova , nova scotia , canada ) and incubated at 25c . the control group consisted of 5  ml of m9 broth with no carbon source ; the experimental group included a 5  ml of m9 broth with sucralose as the only carbon source . turbidity of the cultures was measured over the next 9 days at the same time each day using a sequoia turner ultraviolet light spectrophotometer set to 620  nm wavelength ( figure 2 ) . each bacterial isolate was spread plated into a lawn of confluent growth onto tsa media . filter disks were prepared by whole punching out whatman grade number 2v filter paper and impregnating the disks with 1.6  m sucralose . the antibiotic sensor disk was placed onto the surface of the media , 3 disks per petri dish . the glucose/100  mm sucralose m9 agar completely inhibited growth for 22 of the 28 isolates ( results not shown ) . the isolates that were chosen for gene sequencing and further experimentation all were able to withstand the m9 sucralose and glucose agar ( table 3 ) . of the 28 isolates extracted from environmental samples , only 6 had growth on the sucralose / glucose media . i_61 , rhizobium borbori , citrobacter murlinlae , ensifer arboris , and streptomyces badius . these 6 isolates had fewer cfus on the sucralose / glucose media than they had on the positive control groups of sucrose and glucose and had no growth on the sucralose only m9 agar ( table 3 ) . six unique bacterial isolates were obtained , of which four were gram and 2 gram+ ( table 2 ) . the isolates were subcultured in triplicate in the presence of 27.8  mm , 55.78  mm , 83.75  mm , and 111.7  mm sucralose to elucidate effects of sucralose on bacterial growth , with controls consisting of isolates emended with an additional volume of growth medium . a growth curve showed a decrease in growth with those strains receiving sucralose addition compared to the control ( figure 1 ) . < 0.001 ) difference between control groups and experimental strains amended with 83.75  mm and 111.7  mm sucralose , with experimental strains showing decreased growth rate ( figures 2 , 3 , 4 , 5 , 6 , and 7 ) . these results indicate that the addition of sucralose is a growth inhibitor for multiple strains of bacteria . we observed that there was no statistically significant ( p > 0.99 ) difference between control groups and 25.7  mm for our isolates ( figures 2 , 3 , 4 , 5 , 6 , and 7 ) . the addition of low concentrations of sucralose ( 25.7  nm and less ) had no significant effect on growth , whereas higher levels ( 83.75  mm and 111.7  mm ) reduced growth drastically and possibly led to cell death . at 111.7  mm significant inhibition of total cell culture growth ( figures 3 , 4 , 6 , and 7 ) . of those 6 bacterial isolates not completely inhibited by sucralose , two showed significantly decreased growth ( p < 0.05 ) response in the presence of 55.78  mm sucralose ( figures 2 and 3 ) . the negative effect that sucralose had on their growth rates was directly proportional to the concentration of sucralose added to the growth media . on average , the 27.8  mm and 55.78  mm sucralose treatments did not significantly inhibit the growth rates of these isolates or minimally inhibit the isolates in their growth rates . the 83.75  mm and 111.7  mm treatments did have a rather drastic inhibitory effect on bacterial growth across the board . the inhibition exhibited on all isolates was similar to that of previous studies , in which sucralose causes total growth inhibition of oral bacteria in lab mice [ 5 , 7 ] . . regrowth from inhibited zones was tested ; regrowth indicated a bacteriostatic inhibition for each bacterial isolate . this study is limited in its scope , as it is an inquiry of pure science . this is due to current environmental concentrations being in the 300  ng / l and below range . the research is meant to investigate the possibility of growth inhibition occurring in environmental isolates in pure culture . the current environmental concentrations of sucralose ( 300  ng / l in waste water and less in fresh waters ) do not seem to have any effect on bacterial growth . sucralose is , however , increasing in its concentration due to its inability to be degraded by ph and temperature changes   and its nonmetabolism by microbes ( figure 2 ) . sucralose would at higher concentrations , potentially 55.78  mm , hurt the bacterial community . this type of contamination would take a very long time to accumulate ; however , it is troubling because the bacterial community is the basis for the health of many ecosystems .
sucralose was developed as a low - cost artificial sweetener that is nonmetabolizable and can withstand changes in ph and temperature . 
 it is not degraded by the wastewater treatment process and thus has been found in waste water , estuaries , rivers and the gulf stream . since the molecule can withstand heat , acidification , and microbial degradation , it is accumulating in the environment . 
 the highest concentration of environmental sucralose detected to date is 300  ng / l . 
 our lab has isolated six bacterial species from areas that have been exposed to sucralose . 
 we then cultured these isolates in the presence of sucralose looking for potential sucralose metabolism or growth acceleration . 
 instead we found something very interesting , bacteriostatic effects exhibited on all six isolates . 
 this inhibition was directly proportional to the concentration of sucralose exposure . 
 the efficiency of the growth inhibition seemed to be species specific , with various concentrations inhibiting each organism differently .