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Document Type

Thesis - University Access Only

Award Date


Degree Name

Master of Science (MS)

Department / School

Dairy Science

First Advisor

Sanjeev K. Anand


Reverse Osmosis (RO) membranes are increasingly being used throughout the U.S. in the whey processing industry. Membrane fouling has been identified to be a major operational hurdle in the membrane processing of whey. In addition to the deposition of organic matter, bacterial biofilms may also act as membrane fouling agents. The present investigation was conducted to study the development of bacterial biofilms on spiral wound, reverse osmosis (RO) whey concentration membranes employing both cultural and microscopic techniques. In order to standardize the sampling techniques, initial studies were conducted on three RO membranes (2, 4, and 6 months old) by slicing them into 3 parts: feed, middle, and rear. Each part was further divided into outer, middle, and inner layers. The membrane part/layer analysis for viable counts revealed that the biofilm distribution was non-uniform across the membranes. This study helped to conclude that random sampling would be an appropriate way to study the biofilm formation on membranes. Studies were extended to evaluate the biofilm formations on membranes that were 2, 4, 6, 8, 10, 12, and 14 months old. Swab samples were taken from 5 randomly selected membrane pieces from each cartridge, and analyzed for different types of bacteria such as: total plate counts and aerobic spore formers, Lactobacillus spp., Lactococcus spp. , Coliforms and Escherichia coli, Staphylococcus aureus , Pseudomonas spp., and β hemolytic colonies by using selective agars. The final results were presented as log cfu/cm2 The total plate counts on 2 month to 14 months old membranes were observed to be variable, and did not lead to any specific pattern. However, the results confirmed the presence of the multispecies bacterial biofilms on these membranes. The identified bacterial species were: Enterococcus, Staphylococcus, Micrococcus, Streptomyces 1 Corynebacterium , Bacillus , Klebsiella, Aeromonas , Pseudomonas , Escherichia coli, Streptococcus, and Chryseobacterium. On the other hand, swab samples on unused membrane did not show any microbial counts except one or two colonies on few samples. This proves that new membranes are not a significant source of contamination. Fluorescence microscopic studies revealed both live and dead cells as constituents of the biofilm matrices, and showed the presence and non-uniform distribution of EPS on all tested membranes. The scanning electron microscopic (SEM) of new membrane revealed almost no bacterial contaminants. In case of used membranes, the SEM observations revealed the formation of different biofilm structures demonstrating the fact that it is possible for the same membrane to carry biofilm matrices of different stages. Feed whey and whey retentate samples were collected at Oh and 21h of a typical whey concentration cycle. In addition, the CIP final rinse water was collected after the regular CIP. Bacteria were found to in the feed tank during a typical 21 h whey concentration cycle. On an average, 1 log count increase was observed for different organisms from Oh to 21 h. On the other hand, a relatively higher multiplication rate (with almost 2 log increase) was observed in the whey retentate during the 21h cycle. One of the possible reasons for the greater increase in retentate counts could be attributed to the contamination from membrane biofilms. To study the cross contamination of whey retentate by the membrane biofilms, 16SrDNA sequencing was conducted on a β-hemolytic isolates from the 4mo old membrane biofilm and the corresponding whey retentate sample. Results revealed the presence of the β hemolytic Bacillus cereus cells in both membrane biofilm and corresponding whey retentate samples. In addition, the presence of different organisms in CIP rinse water points out to the possibility of the presence of resistant strains. This study provides a quantitative analysis of bacterial constituents of the biofilm consortia isolated from whey concentration membranes that were obtained from active industrial processes. The study also focused on microbial quality of whey. These findings would help optimize membrane sanitation procedures within the dairy processing industry to improve the membrane performance and product quality.

Library of Congress Subject Headings

Membrane separation
Membranes (Technology)
Reverse osmosis


Includes bibliographical references (pages 52-69)



Number of Pages



South Dakota State University


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