Introduction

In a previous trial Du Plessis et al. (2016) showed that bioreactors reduce chemical oxygen demand (COD) and improve clarity in turbid, high-COD winery wastewaters. Aeration for 120 minutes per day improved bioreactor efficiency. The effect of bioreactor aeration on microbial biodiversity is, however, not known. Bioreactors of the type used by Du Plessis et al. (2016) rely mainly on bacteria and fungi to break down waste products in the wastewater. Bacteria break down winery wastes at moderately high pH levels, whereas fungi metabolise wastes under acid conditions. Bacteria and fungi both form mucilaginous films (biofilms) on solid surfaces. To maximise surface area, bioreactor tanks contain inert plastic spheroids (Bioballs) moulded to create very large, wastewater-accessible internal surface areas. Bacteria and fungi also occur in free-floating (planktonic) form. The bacteria and fungi in the biofilms and plankton must be able to process winery wastewaters over wide pH, composition and concentration ranges. The greater the diversity of the microbial population, the more resilient the system will be. In closed systems, such as bioreactors, microbiological activity may be limited by shortage of oxygen.

The present trial, which was carried out in the same bioreactor used by Du Plessis et al. (2016), aimed to investigate the effects of aeration on the diversity of the bacterial and fungal populations in the plankton and biofilms.

Materials and methods

Two adjacent bioreactors were used, each consisting of four 1 000 ℓ capacity plastic tanks, filled to 75% of their capacity with Bioballs (Figure 1). The tanks in each reactor were installed at progressively lower heights and linked with PVC pipes which carried wastewater from the base of each upstream tank to the top of the next downstream tank. Wastewater was introduced into the top of Tank 1 and discharged through an outlet at the base of Tank 4. This outlet was extended up to the level of the top of Tank 4 to prevent the system from draining, except when wastewater was displaced through the tanks by additions of wastewater to Tank 1. Wastewater was periodically obtained, in bulk, from the holding dam of a nearby winery. After filling for the first time, 270 ℓ of wastewater was pumped into the top of Tank 1 each day for a period of eight months. Allowing for the percentage of the usable tank volume that was occupied by the Bioballs this application rate was equivalent to a theoretical retention time of approximately 12 days, or 3 days per tank. One of the bioreactors was aerated for 120 minutes each day. During this time interval ambient air was pumped into the base of each tank through a diffuser. The tanks in the second bioreactor received no aeration. At 12-day intervals, commencing 12 days after filling, samples of wastewater were drawn from the top of each tank, and 10 randomly-selected Bioballs were collected. The Bioballs were rinsed with water to remove residual planktonic microorganisms, then agitated in an ultrasonic bath to remove the biofilms. After centrifuging to concentrate the separate plankton and biofilm samples, the genomic DNA was extracted, purified, and separated on an agarose gel. DNA amplification was carried out using the polymerase chain reaction. Community diversity was determined by automated ribosomal intergenic spacer analysis, and expressed in terms of the Shannon-Weaver index. In this index, values increase with increasing microbial diversity.

Bioremidiation_figure1_thumbnail

FIGURE 1. A: Bioreactor, consisting of four 1 000 ℓ tanks. Arrows indicate the movement of wastewater. B: An individual Bioball. C: Bioballs exposed on the surface of the wastewater.

FIGURE 2. Effect of aeration and position of tank in bioreactor on Shannon-Weaver community diversity index for planktonic and bioflim bacteria and fungi in parallel aerated and non-aerated bioreactors. Data acquired at 12-day intervals over an eight month period. Values are treatment averages.

 

Results and discussion

Microbial diversity increased from Tank 1 to Tank 2 in all categories, except for the aerated planktonic bacteria. This result was probably anomalous (Figure 2). Planktonic and biofilm bacterial diversities, both aerated and non-aerated, tended to be progressively higher in Tank 3 and 4 than in Tank 2. Conditions in Tank 3 and 4 were thus more conducive to increased bacterial diversity than those in Tank 1 and 2, probably because conditions were more stable, and water quality higher, due to previous processing in Tank 1 and 2.

Fungal diversities in the biofilms were lower than the planktonic bacterial diversities in all four tanks, and did not differ greatly between Tank 2, 3 and 4.

Planktonic fungal diversities were nevertheless lower in Tank 3 and 4 than in Tank 2. This was most notably the case for the planktonic fungi in Tank 4 of the aerated bioreactor.

Averaged over Tank 3 and 4, the effects of aeration on diversity were near zero for the biofilm bacteria and fungi, slightly positive (6.3%) for the planktonic bacteria, and negative (9.5%) for the planktonic fungi. This latter figure is misleading since, as shown in Figure 1, planktonic fungal diversities in aerated Tank 1, 2 and 3 were higher than in the non-aerated tanks.

The generally low diversity indexes in Tank 1 were attributed to turbulence during filling, and to changes in acidity and in the availability of easily decomposable nitrogen and carbon-bearing substrates as the raw wastewater introduced into Tank 1 was progressively acted upon by microorganisms in the subsequent tanks.

These findings indicate that although diversity tended to increase from tank to tank (except for planktonic fungi in which diversity declined), the positive effects of aeration, where present, were small. Conversely, aeration exacerbated the observed decline in planktonic fungal diversity.

Conclusions and recommendations

Under the prevailing trial conditions, aeration, which was shown to increase bioreactor effectiveness in reducing COD and improving turbidity in a previous trial, had a small positive effect on planktonic bacterial diversity, no effect on bacterial and fungal diversity in biofilms, and suppressed planktonic fungal diversity, compared with the non-aerated bioreactor. However, this suppression was only observed in Tank 4. From a practical viewpoint, any disadvantage that may stem from an aeration-induced decline in fungal diversity is likely to be outweighed by the greater COD reducing, and turbidity improving, capabilities of aerated, compared with non-aerated bioreactors.

Summary

The effects of microbiological processing of winery wastewater on bacterial and fungal diversity in plankton and biofilms were determined in aerated and non-aerated bioreactors. Aeration had little effect on bacterial and fungal diversity in biofilms, but slightly suppressed planktonic fungal biodiversity.

Acknowledgements

This project was funded by Winetech, and by the Agricultural Research Council. The authors thank Prof. Karin Jacobs (Stellenbosch University) for inputs on the Shannon-Weaver indexes, Isabella van Huyssteen for processing the data and, especially, Dr. Wesaal Khan for advice concerning the construction of the bioreactor.

Literature cited

Du Plessis, H.R., Wooldridge J., Mulidzi, R., 2016. Bioremediation – a method for reducing chemical oxygen demand and turbidity in winery wastewater. WineLand, May 2016, 71 – 73.

– For more information, contact Reckson Mulidzi at mulidzir@arc.agric.za.

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