In this the chapter, research showed the previously unknown bacterial community of a dairy wastewater system. The hypothesis of mesophilic dominance was proven to be correct with Gram-negative bacteria making up most of the population. Of the culturable bacteria, only one isolate was a Gram-positive bacterium B.cereus. The NGS results showed that the class of bacteria (bacilli) that B.cereus belongs to was present in the dairy wastewater system; however, B.cereus itself was not found. Enterobacteriaceae was the dominant family that was present in the fresh wastewater sample, which was also the dominant family of the bacteria found by the culturing techniques used.
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Seven bacteria in the fresh samples were strong biofilm formers. These consisted of mainly Klebsiella spp (four of seven) with other strong formers consisting of Citrobacter, Enterobacter and Pseudomonas. The culturable bacteria isolated from the extreme biofilm consisted of similar bacteria to the fresh sample with one unique bacteria, Raoultella spp, also noted in the NGS profile. This bacterial species was the strongest biofilm former of the extreme biofilm isolates and therefore could have played a key role in the extreme biofilm formation.
While the biofilm formation of the extreme biofilm isolates was less than that of the fresh samples, due to the unique bacteria and the similar culturable bacteria, it was decided to take the four bacteria from the extreme biofilm for further analysis and modelling of biofilm formation. In the microtiter tests performed so far, biofilm formation representing an extreme biofilm was not seen. The wastewater at the time of formation could have had different characteristics (nutrient level or ion content) than that present at the time of sampling. Therefore, the effect of external factors such as nutrient level temperature and ion content will be investigated.
3.5.1
Future work
This study was a snapshot in that samples were taken at one point in time across five locations. The culturable population was determined from four samples (fresh biofilm, three irrigator wastewater samples) across the wastewater system as well as on the extreme biofilm that started the study, while NGS (DAF sample) was used to analyse the total microbial population entering the wastewater system.
Laboratory trials, inoculated with a combination of the four dominant bacteria C. freundii, C.werkmanii, Enterobacter spp and Raoultella spp will enable a study of the effect of different environments influencing biofilm formation in dairy wastewater systems. While it is unknown whether Raoultella influences the extreme biofilm formation, this was the only unique bacterium isolated from frozen biofilm samples, compared to the fresh samples taken from the wastewater system.
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4
The effect of varying nutrients, temperature, and ion
content on the biofilm formation of bacteria from dairy
plant wastewater
4.1
Abstract
The composition and environmental conditions of the dairy wastewater influence are likely to influence microbial growth in the irrigation system. In the system investigated, the extreme biofilm only occurred intermittently, therefore it was hypothesised that changing environmental conditions is a likely explanation. Manipulating these conditions may assist the dairy industry in controlling biofilm growth in their wastewater irrigation systems.
All four bacteria isolated from the extreme biofilm, are common contaminants found in dairy wastewater. The bacteria were tested as single isolates and as a mixture for their ability to form biofilms under a range of environmental conditions (nutrients, Ca2+ Na+, K+ and temperature). Biofilm formation increased with low nutrients (3g/L Tryptic Soya Broth and the presence of Ca2+ ions (<0.1M). The incubation temperature (15-40°C) effected the biofilm formation of the individual isolates DN1, DN5 and DN7 with optimal biofilm formation at 37°C. DN3, however, formed biofilm at all temperatures and influenced the results from the mixed populations. This appears to be a robust bacterium that is of potential importance in the formation of the extreme biofilm.
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4.2
Introduction
Wastewater composition from dairy plants varies over time depending on the types of products processed, raw milk composition and cleaning chemicals used. However, the effect of the nutrients present in the dairy wastewater on the biofilm forming capability of the bacteria is unknown. The primary treated dairy wastewater stream is routinely tested for: Chemical Oxygen Demand (COD), Biological Oxygen Demand (BOD), pH, temperature, Ca2+, Na+, Mg2+, K+, Total Kjeldahl Nitrogen (TKN), nitrogen (NO3-N, TN, NO2, NH3) and Total Suspended Solids (TSS). Somerton, et al. (2015) showed that 2 mM CaCl2 significantly increased (P ≤ 0.05) the biofilm formation of a dairy derived Geobacillus spp while increased Na+ levels cause a disruption to the biofilm. Assessment of the individual isolates showed individual bacterial responses to these nutrients. However, in the dairy wastewater system these bacteria could potentially work together to protect susceptible bacteria from outside sources to compete for nutrients (Rendueles & Ghigo, 2015). In a primary treated dairy wastewater irrigation system. An extreme biofilm occurred completely blocking the nozzles of the irrigation system. Potential interactions of nutrients could have taken place causing the greater biofilm formation or could potentially disrupt the biofilm matrix in the future. This led to an investigation of the effect of nutrient level (in this case varying TSB concentrations), temperature and ions on biofilm formation in a mixed culture factorial experiment.