Subramanya Rao’s research while affiliated with The University of Hong Kong and other places

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Publications (5)


Exploring the Optimization of Aerobic Food Waste Digestion Efficiency through the Engineering of Functional Biofilm Bio-carriers
  • Article

September 2021

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91 Reads

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4 Citations

Bioresource Technology

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Subramanya Rao

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The possibility of breaking down cellulose-rich food waste through biofilm engineering was investigated. Six previously isolated strains from naturally degrading fruits and vegetables, screened for biofilm-forming ability and cellulolytic activity, were selected to enrich a biocarrier seeding microbial consortium. The food waste model used in this study was cabbage which was aerobically digested under repeated water rinsing and regular effluent drainage. The engineered biocarrier biofilm’s functionality was evaluated by tracing microbial succession following metagenomic sequencing, quantitative PCR, scanning electron microscopy, and cellulolytic activity before and after the digestion processes. The engineered microbial consortium demonstrated superior biofilm-forming ability on biocarriers than the original microbial consortium and generally displayed a higher cellulolytic activity. The presented study provides one of the few studies of food waste aerobic digestion using engineered biofilms. Insights presented in this study could help further optimize aerobic food waste digestion.


Fig. 1. Relative species-level abundance of bacterial pathogens in samples from wooden cutting boards used for different food groups: a) pork, b) poultry, and c) seafood.
Fig. 1. (continued).
Fig. 2. Phylogenetic analysis using Maximum likelihood method for Bacterial pathogens in samples from wooden cutting boards used for different food groups: a) pork, b) poultry, and c) seafood. Blue areas depict Firmicutes (phylum)-clade; red areas, Proteobacteria (phylum)-clade; and cyan areas, Campylobacter (genus)-clade. The green text represents environmental NCBI reference sequences, whereas red text portrays human associated clinical reference sequences. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. (continued).
Comparison of Human/ Environmental association of Bacterial species on wooden cutting board surface used for poultry meat processing in Hong Kong wet markets.

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Questioning the source of identified non-foodborne pathogens from food-contact wooden surfaces used in Hong Kong's urban wet markets
  • Article
  • Full-text available

August 2021

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111 Reads

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7 Citations

One Health

In this study, a phylogenic analysis was performed on pathogens previously identified in Hong Kong wet markets' cutting boards. Phylogenetic comparisons were made between phylotypes obtained in this study and environmental and clinical phylotypes for establishing the possible origin of selected bacterial species isolated from wet market cutting board ecosystems. The results reveal a strong relationship between wet market bacterial assemblages and environmental and clinically relevant phylotypes. However, our poor knowledge of potential cross-contamination sources within these wet markets is further exacerbated by failing to determine the exact or presumed origin of its identified pathogens. In this study, several clinically relevant bacterial pathogens such as Klebsiella pneumoniae, Streptococcus suis and Streptococcus porcinus were linked to cutting boards associated with pork; Campylobacter fetus, Staphylococcus aureus, Escherichia coli, and A. caviae in those associated with poultry; and Streptococcus varanii, A. caviae, Vibrio fluvialis, and Vibrio parahaemolyticus in those associated with seafood. Identifying non-foodborne clinically relevant pathogens in wet market cutting boards in this study confirms the need for safety approaches for wet market meat, including cold storage. The presented study justifies the need for future systematic epidemiological studies to determine identified microbial pathogens. Such studies should bring about significant improvements in the management of hygienic practices in Hong Kong's wet markets and work towards a One Health goal by recognizing the importance of wet markets as areas interconnecting food processing with animal and clinical environments.

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FIGURE 2 | Analysis of the active microbiome in freshwater benthic biofilms subjected to different RNA extraction methods. (A) EggNOG level 1 relative abundance of biofilm communities at the gene expression level for each extraction group. (B) Heat map depicting the 35 most dominant phyla at the gene expression level. Abbreviations of extraction groups denote, K: Column-based kit isolation; R: RNAzol only group; B: Bead-beating group; L: Lysozyme group; P: Cell disruption bomb; LB: Lysozyme + Bead-beating group.
FIGURE 3 | Non-supervised Orthologous Groups (EggNOG) annotation analysis of samples from freshwater benthic biofilms. (A) Numbers of genes matched to the EggNOG database in the combined column-and organic isolation-based groups. (B) Functional annotation analysis of the relative abundances in the six extraction groups, based on EggNOG database level 1 including 24 taxa. Abbreviations of extraction groups denote, K: Column-based kit isolation; R: RNAzol only group; B: Bead-beating group; L: Lysozyme group; P: Cell disruption bomb; LB: Lysozyme + Bead-beating group.
FIGURE 4 | Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and functional annotation analysis of freshwater benthic biofilm samples. (A) Number of genes annotated to KEGG database at level 2 in the combined column-and organic isolation-based groups. X-axis indicates the number of genes annotated in each KEGG pathway; Y -axis indicates the name of KEGG level 2 pathways. (B) Functional annotation analysis of the relative abundances in the six extraction groups, based on KEGG database level I including 6 pathways. Abbreviations of extraction groups denote, K: Column-based kit isolation; R: RNAzol only group; B: Bead-beating group; L: Lysozyme group; P: Cell disruption bomb; LB: Lysozyme + Bead-beating group.
FIGURE 5 | Carbohydrate-Active Enzymes Database (CAZy) annotation analysis of freshwater benthic freshwater biofilm samples. (A) Numbers of genes annotated to the CAZy database in the combined column-and organic isolation-based groups. (B) Functional annotation analysis of the relative abundances in the six extraction groups, based on CAZy database level 1 including six major function classes. Abbreviations of extraction groups denote, K: Column-based kit isolation; R: RNAzol only group; B: Bead-beating group; L: Lysozyme group; P: Cell disruption bomb; LB: Lysozyme + Bead-beating group.
FIGURE 6 | Clustering analysis of differentially expressed genes (DEGs). (A) Hierarchical clustering graph of the 12086 DEGs based on the average log 10 (FPKM + 1) values of all genes in each cluster. Red bands indicate higher gene expression, while blue bands indicate lower gene expression. (B) Clustering of the 12086 DEGs into eight subclusters. The number of genes in each cluster is presented at the top of each cluster. Blue lines indicate the average relative expression levels in each subcluster relative to a log2 ratio of 0, which is represented by the red line. Gray lines represent the relative expression levels of each gene in each cluster. The sample names and corrected expression levels are presented on the x axis and y axis, respectively.
Active Microbiome Structure and Functional Analyses of Freshwater Benthic Biofilm Samples Influenced by RNA Extraction Methods

March 2021

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524 Reads

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3 Citations

Advances in high-throughput sequencing technologies have enabled extensive studies of freshwater biofilms and significant breakthroughs in biofilm meta-omics. To date, however, no standardized protocols have been developed for the effective isolation of RNA from freshwater benthic biofilms. In this study, we compared column-based kit RNA extraction with five RNAzol-based extractions, differentiated by various protocol modifications. The RNA products were then evaluated to determine their integrity, purity and yield and were subjected to meta-transcriptomic sequencing and analysis. Significant discrepancies in the relative abundance of active communities and structures of eukaryotic, bacterial, archaebacterial, and viral communities were observed as direct outcomes of the tested RNA extraction methods. The column isolation-based group was characterized by the highest relative abundance of Archaea and Eukaryota, while the organic isolation-based groups commonly had the highest relative abundances of Prokaryota (bacteria). Kit extraction methods provided the best outcomes in terms of high-quality RNA yield and integrity. However, these methods were deemed questionable for studies of active bacterial communities and may contribute a significant degree of bias to the interpretation of downstream meta-transcriptomic analyses.


Valorization of volatile fatty acids from the dark fermentation waste Streams-A promising pathway for a biorefinery concept

March 2021

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210 Reads

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76 Citations

Renewable and Sustainable Energy Reviews

In recent years, much attention has been directed towards the integration of dark fermentation process into a biorefinery concept to enhance the energetic gains, thereby improving the competitiveness of this process. The volatile fatty acids (VFAs) from dark fermentative H2-producing processes serve as precursors for the microbial synthesis of a broad spectrum of biotechnologically-important products such as biofuels and biocommodities. These products are desirable substrates for secondary bioprocesses due to their biodegradable nature and affordability. This short review discusses the use of acidogenic-derived VFAs in the production of value-added compounds such as polyhydroxyalkanoates (PHAs) alongside the microbial-based fuels (hydrogen, biogas, and electricity), and other valuable compounds (succinic acid, citric acid, and butanol). The review also highlights the strategies that have been used to enhance the extraction of VFAs from acidogenic effluents and other related waste streams. The application of novel enhancement techniques such as nanoparticles during VFAs recovery is also discussed in this work. Furthermore, the work highlights some of the recent advances in dark fermentation-based biorefinery, particularly the development of pilot-scale processes. Finally, the review provides some suggestions on the advancement of dark fermentation-based biorefineries using VFAs that are derived from acidogenic processes.


Figure 1. (a) Selected wet markets in Hong Kong based on their modern vs. traditional profiles; (b) Multidimensional scaling plot (MDS) of all samples from wooden cutting boards used for different food groups (pork, poultry, and seafood). (c) MDS plot for (i) modern wet market (left panel) and (ii) traditional wet market (right panel). (d) MDS plot for (i) uncleaned (left panel) and (ii) cleaned (right panel) cutting boards from different wet markets.
Bacterial species on wooden cutting board surfaces in Hong Kong wet markets.
Impacts of Wet Market Modernization Levels and Hygiene Practices on the Microbiome and Microbial Safety of Wooden Cutting Boards in Hong Kong

December 2020

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687 Reads

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14 Citations

Microorganisms

Accessing food through wet markets is a common global daily occurrence, where fresh meat can be purchased to support an urbanizing world population. Similar to the wet markets in many other metropolitan cities in Asia, Hong Kong wet markets vary and are characterized by differing hygiene routines and access to essential modern technologies. The lack of risk assessments of food contact surfaces in these markets has led to substantial gaps in food safety knowledge and information that could help improve and maintain public health. Microbial profiling analyses were conducted on cutting boards that had been used to process pork, poultry, and seafood at 11 different wet markets. The markets differed in hygiene protocols and access to modern facilities. Irrespective of whether wet markets have access of modern infrastructure, the hygiene practices were largely found to be inefficient based on the prevalence of bacterial species typically associated with foodborne pathogens such as Campylobacter fetus, Clostridium perfringens, Staphylococcus aureus, and Vibrio parahaemolyticus; indicator organisms such as Escherichia coli; as well as nonfoodborne pathogenic bacterial species potentially associated with nosocomial infections, such as Klebsiella pneumoniae and Enterobacter cloacae. Other Vibrio species, V. parahaemolyticus and V. vulnificus, typically associated with contaminated raw or undercooked seafood with the potential to cause illness in humans, were also found on wooden cutting boards. This study indicated that the hygienic practices used in Hong Kong wet markets are not sufficient for preventing the establishment of spoilage or pathogenic organisms. This study serves as a basis to review current hygiene practices in wet markets and provides a framework to reassess existing safety protocols.

Citations (5)


... Additionally, to observe the optimum time for the organic compounds to be degraded by the biofilm, the soluble SCOD was also measured by the OFAT process, where 328 mg of biofilm were chosen as the optimum biofilm amount based on the TCOD analysis. Fung et al. [49] have noticed that the biodegradability of FW was characterized by a decrease in the TCOD value, as found in this experiment. ...

Reference:

Hydrolysis of Food Waste with Immobilized Biofilm as a Pretreatment Method for the Enhancement of Biogas Production
Exploring the Optimization of Aerobic Food Waste Digestion Efficiency through the Engineering of Functional Biofilm Bio-carriers
  • Citing Article
  • September 2021

Bioresource Technology

... However, efforts crucial to reduce losses for securing future sustainability are practically absent. Many previous studies have focused on cutting boards harboring microbes that lead to foodborne diseases and the safety of wood as a meat-contact material (Lucke & Skowvrska 2015;Munir et al. 2019;Sekoai et al. 2020;Rao et al. 2021). Others have examined the performance of vegetable chopping boards from an ergonomic perspective of proper handling and discomfort (Radhika & Lakshmi 2021). ...

Questioning the source of identified non-foodborne pathogens from food-contact wooden surfaces used in Hong Kong's urban wet markets

One Health

... In brief, approximately 1.5 million metric tons of acetic acid were produced globally in 2022, and this number is expected to increase to 2.8 million metric tons by 2030 [46]. Similarly, the demands for butyric acid and propionic acid are also increasing due to their application in various industrial sectors, including biopolymers, bioplastics, animal feed, food, and pharmaceuticals [47]. Generally, VFAs are produced during acidification or fermentation, which follows the hydrolysis stage. ...

Valorization of volatile fatty acids from the dark fermentation waste Streams-A promising pathway for a biorefinery concept
  • Citing Article
  • March 2021

Renewable and Sustainable Energy Reviews

... The mass of the 15 samples in each case was determined by weighing in order to determine the volume of materials used in the study. Before the start of the experiment, the biofilm was removed from the samples by ultrasound [13] and the samples were sterilized with 70% ethyl alcohol, which was removed by washing the samples in sterile distilled water before applying them to the soil. The samples were mixed with the soil in sterile polypropylene containers, each of 120 mL and with 5 holes of 1 mm diameter at the bottom. ...

Active Microbiome Structure and Functional Analyses of Freshwater Benthic Biofilm Samples Influenced by RNA Extraction Methods

... In this survey, 39.6% of samples among a collection of 660 hot spot swabs from various surfaces (door knobs, cutting knives, drinking water pots, food plates, and leftover foods) tested positive for V. cholerae. To our knowledge, the only published study is that of Ngan et al. (2020), who analyzed the microbial profiles of biofilms on wooden cutting boards from 11 wet markets in Hong Kong and found natural surface presence of V. parahaemolyticus and V. vulnificus, which was later confirmed by Rao et al. (2021). Cutting boards represent hotspots for cross-contamination ranging from food processing plants to the kitchens of consumers. ...

Impacts of Wet Market Modernization Levels and Hygiene Practices on the Microbiome and Microbial Safety of Wooden Cutting Boards in Hong Kong

Microorganisms