Research Methodology

Advanced approaches for sustainable aquaculture development in Qatar

Methodological Framework

The methodology for achieving the project objectives will utilize comprehensive facilities at HBKU-CSE, HBKU-QEERI, and HBKU-QBRI labs. The project employs a systematic approach with five key objectives, each with specific technical methodologies.

1

Reactor Setup and Continuous Operation

Implementation of continuous operation systems for wastewater treatment and biomass production.

Equipment and Setup

155 L flat panel reactor and 60 L anaerobic pilot scale reactor (BIOPAQ IC) housed in a translucent greenhouse equipped with necessary feed and recirculation pumps for continuous operation.
Monitoring equipment: Solar irradiance measured using Black-Comet UV-VIS spectrophotometer (StellarNet Inc); pH and temperature monitored using ThermoFisher Scientific multiparameter meter (Orion Ross Ultra pH/ATC Triode).
Analytical instruments: Optical density assessed using UV-3600 plus UV-VIS-NIR spectrophotometer (Shimadzu).

Chemical Analysis Methods

Sample Preparation: Centrifuging samples at 6,000 g (Sorvall Lynx 6000, ThermoScientific), filtering supernatant through 0.2 µm sterile syringe filters (VWR).
COD Measurement: Closed micro-digestion method using Hach high-range COD vials.
TOC, IC, and TN Analysis: TOC analyzer (TOC-L series with TNM-1, Shimadzu).
VFA and Anion Analysis: Ion chromatography (940 Professional IC Vario, Metrohm) for VFAs and anions such as phosphate.
Biomass Concentration: TSS and VSS measurements using standard methods (APHA, 2012).
Microbial Community Analysis: DNA extraction using DNeasy PowerBiofilm kit (Qiagen) and 16S metagenomics as described in George et al. (2022).
2

Biomass Characterization and Analysis

Comprehensive analysis of biomass composition, nutritional value, and safety parameters.

Sample Preparation

Biomass will be harvested, freeze-dried, and homogenized for analysis.

Analytical Methods

Protein Content: Ultrasonic-assisted alkali extraction followed by quantification with Modified Lowry method as proposed by Wada et al. (2024).
Amino Acids Characterization: Complete enzymatic hydrolysis followed by analysis as described by Wada et al. (2024).
Lipid and Carbohydrate Content: Bligh and Dyer method for lipids, Anthrone method for carbohydrates.
Pigments and Coenzyme-Q10: Spectrophotometric measurement.
Trace Elements and Heavy Metals: Acid microwave digestion followed by ICP-OES analysis as described by Wada et al. (2023).
Elemental Composition: CHNS analysis using elemental analyzer.
Nucleic Acid Quantification: NanoDrop Microvolume UV-Vis 2000 Spectrophotometer.
Endotoxin Levels: Limulus amebocyte lysate (LAL) assay.
Organic Compounds Characterization: Gas chromatography/mass spectrometry following EPA methods 8260 B and 8260 C.
3

Biomass Digestibility Enhancement

Implementation of pretreatment methods to improve biomass digestibility for aquaculture feed.

Pretreatment Methods

Extrusion at high temperatures (>90ºC)
Autoclaving
Alkali and enzymatic treatments
Sonication
Bead milling (previously utilized for biomass cellular disruption study - Wada et al., 2024)

Key Variables Examined

Pretreatment duration
Integrity of cell walls post-treatment

Evaluation Techniques

Physical Characteristics: Scanning electron microscope (SEM, Quanta 650 FEG-ESEM) and transmission electron microscope (TEM, Talos F200C TEM).
Digestibility Assessment: In vitro enzymatic digestibility assays using digestive enzymes (trypsin, pepsin, amylase, maltase, lipase) to mimic gastrointestinal conditions in fish and shrimp.
Protein Solubility: Analytical techniques similar to protein quantification methods.

The two most effective pretreatment methods will be selected for subsequent shrimp feeding trials based on apparent digestibility coefficients.

4

Aquaculture Feeding Trials

Controlled feeding trials to evaluate the performance of PNSB-based feeds.

Shrimp Trials (Litopenaeus vannamei)

Housing: Larvae reared in aerated glass aquariums with 3-4 L artificial seawater
Stocking density: ~600 post larvae per square meter (20 shrimp per aquarium)
Duration: 10-week trial duration with eight test conditions
Feeding: Three times daily with blends of the best two digested PNSB MP groups at replacement levels of 30%, 50%, and 100%
Monitoring: Weekly growth, health, performance (FCR, intake)
Endpoint analysis: Protein, elemental, and proximate composition

Asian Sea Bass Trials

Housing: Juveniles raised in recirculating aquaculture systems (300 L tanks)
Stocking: 20-50 fish per tank (subject to resource availability)
Duration: 12-week trial duration
Feeding: Twice daily with PNSB MP blended feeds at 30%, 50%, 70%, and 100% replacement levels
Monitoring: Initial and weekly measurements of weight and length
Endpoint variables: Similar to shrimp trials
5

Sustainability and Economic Assessment

Comprehensive evaluation of socio-economic viability and environmental sustainability.

Socio-Economic Assessment

Stakeholder Surveys: Semi-structured questionnaires for local fish farmers and feed sellers; online survey for fish-consuming adults in Qatar.
Data Analysis: Quantitative data analyzed using descriptive and inferential statistics with Microsoft Excel and JASP 0.18.1; qualitative data from key informant interviews with government stakeholders undergo thematic analysis.

Techno-Economic Analysis

Comparison of costs for producing a tonne of alternative feed locally versus conventional aquafeed production and importation, utilizing standardized Excel formulas and literature data.

Life Cycle Assessment (LCA)

Approach: Cradle-to-gate approach using Gabi software.
Impact Categories: Climate change, water use, land use, and other environmental impacts using Recipe methodology.
Comparison: Environmental impacts of locally produced feed versus imported conventional feed.